anaerobic soil disinfestation: meta-analysis and

University of Tennessee, Knoxville University of Tennessee, Knoxville

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Anaerobic Soil Disinfestation: Meta-analysis and Optimization of Anaerobic Soil Disinfestation: Meta-analysis and Optimization of

Amendment Carbon Rate and C:N Ratio to Control Key Plant Amendment Carbon Rate and C:N Ratio to Control Key Plant

Pathogens and Weeds Pathogens and Weeds

Utsala Shrestha University of Tennessee, Knoxville, [email protected]

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mailto:[email protected]

To the Graduate Council:

I am submitting herewith a dissertation written by Utsala Shrestha entitled "Anaerobic Soil

Disinfestation: Meta-analysis and Optimization of Amendment Carbon Rate and C:N Ratio to

Control Key Plant Pathogens and Weeds." I have examined the final electronic copy of this

dissertation for form and content and recommend that it be accepted in partial fulfillment of the

requirements for the degree of Doctor of Philosophy, with a major in Plants, Soils, and Insects.

David M. Butler, Major Professor

We have read this dissertation and recommend its acceptance:

Bonnie H. Ownley, Annette L. Wszelaki, Arnold M. Saxton

Accepted for the Council:

Carolyn R. Hodges

Vice Provost and Dean of the Graduate School

(Original signatures are on file with official student records.)

Anaerobic Soil Disinfestation: Meta-analysis and Optimization of Amendment

Carbon Rate and C:N Ratio to Control Key Plant Pathogens and Weeds

A Dissertation Presented for the

Doctor of Philosophy

Degree

The University of Tennessee, Knoxville

Utsala Shrestha

August 2016

ii

Copyright © 2016 by Utsala Shrestha

All rights reserved.

iii

DEDICATION

In the memory of

Sheela Shrestha

iv

ACKNOWLEDGEMENTS

I would like to acknowledge all those individuals who helped me directly or indirectly during

this dissertation preparation.

It is my great pleasure to express my deep sense of gratitude and sincere appreciation to Dr.

David M. Butler, for his valuable guidance and constant encouragement from the beginning to

the end of my doctorate degree. He always made time for my works and me, and listen to me

patiently. He is a cheerful and gem of person. I equally acknowledge and express my sincere

gratitude to Dr. Bonnie H. Ownley for her constant support and suggestions during my entire

doctoral study. She is one of great mentor in both life and study, and her work inspires me to

follow her path. I am also deeply grateful to Dr. Arnold M. Saxton for his guidance in statistics.

He has a pleasant personality and I will always remember his nice smile. I would also like to

thank Dr. Annette L. Wszelaki for her constructive comments and helpful suggestions for the

completion of this study. I am so glad to have them all in my Advisory Committee.

I equally express my heartfelt thanks to Dr. Robert M. Auge for support and cooperation to write

a meta-analysis chapter/article.

This dissertation would have not been possible without assistance from the members of Dr.

Butler’s lab: Sarah E. Inwood, Heather Toler, Hanna, Sarah, Will, Justin, Cody, Grant, Geoferry,

Zach, and Bronson, members of Dr. Ownley’s lab: Mary Dee, Alex, Sara and staff members of

UT Plateau Research and Education Center, Crossville: John (Walt) Hitch and Ann Moore. I am

also thankful to Dr. Erin Rosskopf and Jason Hong for their support and suggestions.

It is the matter of my immense pleasure to express my deep gratitude and heartfelt respect to my

all-family members (Shrestha and Piya) and friends for their affection, inspirations, and support

to precede my academic carrier.

Last but not the least, thanks to my beloved husband Sarbottam Piya for his constant support

during my dissertation preparation, his constructive feedback, and, support in every step of the

way.

v

ABSTRACT

Anaerobic soil disinfestation (ASD) is an environmentally friendly and cost effective pre-plant

soil treatment technique that allows effective control of soilborne pests by creating anaerobic

conditions, particularly for specialty and organic crop production under diverse environmental

conditions. In spite of being a proven technique, ASD has to be optimized to fit into local

production systems with specific pathogen pressure using locally available amendments for

successful implementation on a commercial scale. Our meta-analysis study on soilborne

pathogens, plant parasitic nematodes, and weeds validated that ASD is an effective approach to

control various soilborne pathogens. This study aims to optimize the carbon rate and carbon to

nitrogen ratio (C:N) of two ASD amendments namely, dry molasses and wheat bran to suppress

Fusarium oxysporum, Sclerotium rolfsii and Cyperus esculentus tubers for a moderate soil

temperature regime. Evaluation of survivability of recovered tubers, Fusarium oxysporum and

Sclerotium rolfsii inocula corroborated with the finding of our meta-analysis that ASD

effectively promotes tuber and pathogen propagule mortality. Evaluation of various carbon

amendment rates maintained at a C:N ratio of 30:1 showed that 4 milligrams of carbon per gram

of soil was the most effective to induce sclerotial mortality and parasitism. We found that

maintaining an amendment C:N ratio within the range of 20:1 to 30:1, with carbon rate at 4

milligrams of carbon per gram of soil, is effective in generating favorable anaerobic conditions

resulting in higher pathogen suppression and enhancement of beneficial mycoparasites.

Keywords: Anaerobic / biological soil disinfestation, beneficial microorganisms, Fusarium

oxysporum, meta-analysis, mycoparasitism, Sclerotium rolfsii, soilborne pathogens,

Trichoderma, yellow nutsedge

vi

TABLE OF CONTENTS

Introduction .............................................................................................................. 1

Appendix ..................................................................................................................................... 4

Chapter 1 A meta-analysis of the impact of anaerobic soil disinfestation

on pest suppression and yield of horticultural crops ............................................ 5

Abstract ....................................................................................................................................... 7

1. Introduction ............................................................................................................................. 8

2. Materials and Methods .......................................................................................................... 10

2.1 Data Collection ............................................................................................................... 10

2.2 Moderator variables ....................................................................................................... 11

2.3 Effect size and meta-analysis .......................................................................................... 12

2.4 Publication bias and sensitivity analysis ........................................................................ 13

3. Results ................................................................................................................................... 14

3.1 Measure of efficacy ......................................................................................................... 16

3.2 Pathogens ........................................................................................................................ 17

3.2.1 Experimental conditions for pathogen studies ............................................................. 18

3.2.2 Amendment effect on pathogen suppression ................................................................ 18

3.3 Nematode suppression .................................................................................................... 19

3.4 Weed suppression............................................................................................................ 20

3.5 Yield ................................................................................................................................ 21

4. Discussion ............................................................................................................................. 22

4.1 Is ASD effective for pathogen suppression? ................................................................... 22

4.2 Conditions favoring ASD effectiveness on pathogen suppression .................................. 23

4.3 Contribution of ASD amendments to pathogen suppression .......................................... 25

4.5 ASD effect on nematode suppression .............................................................................. 26

4.6 ASD effect on weed suppression ..................................................................................... 27

4.7 ASD effect on crop yield ................................................................................................. 27

5. Conclusion ............................................................................................................................ 28

6. Acknowledgements ............................................................................................................... 29

vii

References ................................................................................................................................. 30

Appendix ................................................................................................................................... 38

Chapter 2 Effect of anaerobic soil disinfestation amendment C:N ratio

on tuber germination, growth and reproduction of yellow nutsedge

(Cyperus esculentus) and organic acid production ............................................. 53

Abstract ..................................................................................................................................... 55

1. Introduction ........................................................................................................................... 57


2.1 Experimental setup.......................................................................................................... 60

2.2 Experimental procedure.................................................................................................. 61

2.3 Cumulative redox potential ............................................................................................. 61

2.4 Soil properties ................................................................................................................. 61

2.5 Yellow nutsedge survival assessment .............................................................................. 62

2.6 Organic acid assay ......................................................................................................... 62

2.7 Statistical analysis .......................................................................................................... 63

3. Results and Discussion ......................................................................................................... 63

3.1 Soil temperature and moisture ........................................................................................ 63

3.2 Cumulative anaerobic activity and soil pH .................................................................... 64

3.3 Soil nutrients and C:N ratio ............................................................................................ 65

3.4 Tuber germination .......................................................................................................... 66

3.5 Nutsedge growth and reproduction ................................................................................ 67

3.6 Organic acid assay ......................................................................................................... 68

4. Conclusion ............................................................................................................................ 69

References ................................................................................................................................. 70

Appendix ................................................................................................................................... 78

Chapter 3 Organic amendment type and C:N ratio impact on Fusarium

oxysporum f. sp. lycopersici following anaerobic soil disinfestation .................. 89

Abstract ..................................................................................................................................... 91

1. Introduction ........................................................................................................................... 92

viii


2.1 Fusarium inoculum preparation ..................................................................................... 94

2.3 Pot experiment ................................................................................................................ 95

2.4 Assessment of Fusarium population ............................................................................... 96

2.5 Field experiment ............................................................................................................. 96

2.5.1 Layout treatment establishment ................................................................................... 96

2.5.2 Anaerobic soil conditions and Fusarium assay ........................................................... 97

2.5.3 Crop performance and root disease assessment .......................................................... 98

2.6 Statistical analysis .......................................................................................................... 98

3. Results ................................................................................................................................... 99

3.1 Pot study.......................................................................................................................... 99

3.1.1 Gravimetric soil moisture, soil temperature, and soil pH ........................................... 99

3.1.2 Anaerobic soil conditions .......................................................................................... 100

3.1.3 Fusarium population .................................................................................................. 100

3.2 Field study ..................................................................................................................... 100

3.2.1 Anaerobic soil conditions .......................................................................................... 101

3.2.2 Fusarium population .................................................................................................. 101

4. Discussion ........................................................................................................................... 102

5. Conclusion .......................................................................................................................... 106

References ............................................................................................................................... 107

Appendix ................................................................................................................................. 112

Chapter 4 Effects of amendment C:N ratio and carbon rate on

germination and parasitism of sclerotia of Sclerotium rolfsii following

anaerobic soil disinfestation ................................................................................125

Abstract ................................................................................................................................... 127

1. Introduction ......................................................................................................................... 128

2. Materials and Methods ........................................................................................................ 130

2.1 Sclerotia production and inoculum preparation........................................................... 130

2.2 Growth chamber study .................................................................................................. 131

2.2.1 Amendment and C:N ratio effect................................................................................ 131

ix

2.2.2 Amendment C rate effect ............................................................................................ 131

2.3 Field study ..................................................................................................................... 132

2.4 Assessment of sclerotia following ASD treatment ........................................................ 132

2.5 Measuring soil moisture, temperature, pH, and anaerobicity ...................................... 132

2.6 Data analysis ................................................................................................................ 133

3. Results ................................................................................................................................. 133

3.1 Growth chamber examination of ASD amendment and C:N ratio effect...................... 133

3.1.1 Soil characteristics and anaerobic conditions ........................................................... 134

3.1.2 Sclerotial germination and parasitism ....................................................................... 134

3.2 Growth chamber examination of ASD amendment C rate ............................................ 135

3.2.1 Soil characteristics and anaerobic conditions ........................................................... 135

3.2.2 Sclerotial germination and parasitism ....................................................................... 136

3.3 Field examination of ASD effect on anaerobic conditions, sclerotial germination and

parasitism ............................................................................................................................ 137

4. Discussion ........................................................................................................................... 138

5. Conclusion .......................................................................................................................... 140

References ............................................................................................................................... 141

Appendix ................................................................................................................................. 146

Chapter 5 Assessment of beneficial microorganisms: Trichoderma,

actinomycetes, Bacillus and root colonizers in anaerobic soil

disinfestation. ........................................................................................................157

Abstract ................................................................................................................................... 159

1. Introduction ......................................................................................................................... 160

2. Material and Methods ......................................................................................................... 162

2.1 Sclerotia inoculum preparation .................................................................................... 162

2.2 Isolation and spore suspension of Trichoderma asperellum ........................................ 163

2.3 ASD treatments and/or biocontrol treatments .............................................................. 163

2.4 Pot setup........................................................................................................................ 163

2.5 Pathogenicity testing ..................................................................................................... 164

2.6 Assessment of sclerotial germination and parasitism ................................................... 165

x

2.7 Quantification of Trichoderma, actinomycetes, and Bacillus from soil ....................... 165

2.8 Molecular identification ................................................................................................ 166

2.9 ASD effect on AM fungi................................................................................................. 167

2.10 Data analysis .............................................................................................................. 167

3. Results ................................................................................................................................. 167

3.1 Germination of sclerotia ............................................................................................... 168

3.2 Sclerotial parasitism ..................................................................................................... 169

3.4 Effects on beneficial organisms .................................................................................... 171

3.5 Effect on nodule and colonization of AM ...................................................................... 171

3.6 Effect on root and shoot biomass .................................................................................. 171

4. Discussion ........................................................................................................................... 172

5. Conclusion .......................................................................................................................... 174

6. Acknowledgements ............................................................................................................. 174

References ............................................................................................................................... 175

Appendix ................................................................................................................................. 181

Summary ...............................................................................................................199

Vita ........................................................................................................................202

xi

LIST OF TABLES

Table 1-1. Number of studies by country and USA states and response variables

examined ..................................................................................................................................38

Table 1-2. Levels and attributes within each categorical moderator variable tested

for significance of pest suppression and yield responses .........................................................39

Table 1-3. Measures used in characterizing publication bias for each effect size

(after Borenstein, 2005) ...........................................................................................................41

Table 2-1. Amount of C amendments for different C:N ratios at 4 mg C g-1

of soil

for each pot ..............................................................................................................................78

Table 2-2. Soil inorganic phosphorus, soil nitrite+nitrate-N (NO2+NO3-N) and

ammonium-nitrogen (NH4-N) and total inorganic N as affected by soil

amendments and amendment C:N ratios .................................................................................79

Table 2-3. Tuber ratings of non-germinated tubers for amendment and C:N ratio

treatments .................................................................................................................................80

Table 3-1. Gravimetric soil moisture content (g g-1

) of soil after 3 weeks of ASD

treatment, pot study ................................................................................................................112

Table 3-2. Effect of location of Fusarium inoculum packet in the pot on Fusarium

populations, pot assays...........................................................................................................113

Table 3-3. Effect of C: N ratio soil treatment on soil gravimetric moisture content

(g g-1

) in field condition .........................................................................................................114

Table 3-4. Effect of C: N ratio soil treatment on endemic soil Fusarium

populations in field assays .....................................................................................................115

Table 3-5. Growth characteristics of 8-week-old tomato plant transplanted at 5

weeks old in pots after termination of ASD treatment (Trial 1) ............................................116


weeks old in pots after termination of ASD treatment (Trial 2) ............................................117

Table 4-1. Treatment: carbon to nitrogen ratio, carbon amendments, and

amendments rates ...................................................................................................................146

xii

Table 4-2. Mean gravimetric soil moisture, soil pH and anaerobic condition

during treatment and post treatment from C:N ratio pot assay conducted in the

growth chamber .....................................................................................................................147

Table 4-3. Effect of carbon soil treatment on sclerotia of Sclerotium rolfsii in pot

assay, C:N ratio growth chamber study, 2013 .......................................................................148


weeks after termination of ASD treatment sclerotia inoculated pots, carbon

rate growth chamber study .....................................................................................................149

Table 4-5. Effect of C: N ratio soil treatment on soil gravimetric moisture content

(g g-1

) in field conditions ........................................................................................................150

Table 5-1. List of biocontrol agents and ASD treatment .............................................................181

Table 5-2. Correlation among sclerotial germination, sclerotial parasitism and soil

properties during ASD treatment, pot study, 2015 ................................................................182

xiii

LIST OF FIGURES

Figure A-1. Map of countries/states (in black) conducting ASD study .......................................... 4

Figure 1-1. Flow diagram showing the study selection procedure ................................................42

Figure 1-2. Weighted summary effect sizes (lnR) and 95% confidence intervals

(CIs) for ASD moderator ‘measure of efficacy’ (various measures of pathogen

growth and survival used in the literature). .............................................................................43


(CIs) for ASD effect on suppression of pathogens and beneficial

mycoparasites. ..........................................................................................................................44


(CIs) for ASD effect on pathogen suppression under various experimental

conditions. ................................................................................................................................45


(CIs) for ASD amendment effect on pathogen suppression. ...................................................46


(CIs) for ASD effect on nematode suppression. ......................................................................47


(CIs) for ASD effect on weed suppression. .............................................................................49


(CIs) for ASD effect on yield response. ...................................................................................51


(CIs) for ASD amendment effect on yield response. ...............................................................52

Figure 2-1. Effect of amendment C:N ratio on mean cumulative anaerobic

condition during ASD. .............................................................................................................81

Figure 2-2. Effect of amendment C:N ratio on mean soil pH in CaCl2 at treatment

termination. ..............................................................................................................................82

Figure 2-3. Effect of amendment and amendment C:N ratio on mean percentage

of tuber germination per pot during ASD. ...............................................................................83


of number of tuber production per pot during ASD. ...............................................................84

xiv


of dry shoot biomass production per pot during ASD. ............................................................85


of dry root biomass production per pot during ASD. ..............................................................86

Figure 2-7. Total organic acids present in soil at 7 and 14 days post treatment

initiation. ..................................................................................................................................87

Figure 2-8. Various organic acids present in soil at 7 and 14 days post treatment

initiation. ..................................................................................................................................88

Figure 3-1. Effect of amendment C:N ratio on soil pH at treatment termination,

pot study. ................................................................................................................................118

Figure 3-2. Effect of amendment C:N ratio on cumulative anaerobic condition

during ASD, pot study. ..........................................................................................................119

Figure 3-3. Effect of amendment and amendment C:N ratio on Fusarium

populations during ASD treatment, pot study. .......................................................................120

Figure 3-4. Effect of amendment C:N ratio on percentage of iron oxyhydroxide

paint removal following ASD treatment, field study, 2013-2014. ........................................121

Figure 3-5. Effect of amendment C:N ratio on Fusarium oxysporum inoculum

populations following ASD treatment, field study, 2013-2014. ............................................122

Figure 3-6. Mean pepper fruit numbers ha-1

harvested for the year 2013 and 2014.

i. total fruit, ii. marketable fruit (Fancy+US No.1 + US No.2), iii. Fancy and

iv. culled fruit .........................................................................................................................123

Figure 3-7. Mean pepper yield (mt ha-1

) for the year 2013 and 2014. i. total yield,

ii. marketable yield (Fancy + US No.1 + US No.2), iii. Fancy and iv. culled

yield........................................................................................................................................124

Figure 4-1.Effect of amendment C:N ratio on soil pH (A), cumulative anaerobic

condition (B), percentage sclerotial germination (C), and percentage

decomposed sclerotia (D) during ASD treatment, pot study. ................................................151

Figure 4-2. Effect of amendment C:N ratio on percentage sclerotial parasitism by

Trichoderma (A), Fusarium (B), other fungi (C), and Fusarium and other

fungi (D) after ASD treatment, pot study. .............................................................................152

xv

Figure 4-3. Effect of carbon amendments at depths 5-cm (top) and 15-cm

(bottom) on percentage sclerotial germination (A), percentage sclerotial

parasitism by Trichoderma (B), Fusarium (C), and other fungi (D) after ASD

treatment, pot study. ...............................................................................................................153

Figure 4-4. Effect of amendment C rates on soil pH (A), cumulative anaerobic

condition (B), percentage sclerotial germination (C), and percentage

Trichoderma parasitism (D) during ASD treatment, pot study. ............................................154

Figure 4-5. Effect of amendment C rates on percentage sclerotial parasitism in pot

trial 1 (A) and trial 2 (B). .......................................................................................................155

Figure 4-6. Effect of dry molasses amendment at different C:N ratios on

percentage sclerotial germination (A), percentage sclerotial parasitism by

Trichoderma (B) in the year 2013 (field assay 1) and 2014 (field assay 2);

percentage sclerotial parasitism other than Trichoderma in year 2013 (C) and

2014 (D) after ASD treatment................................................................................................156

Figure 5-1. Pot set-up in the growth chamber for ASD study on beneficial

organism. ................................................................................................................................183

Figure 5-2. Process of soil serial dilutions to measure actinomycetes, Bacillus and

Trichoderma populations. ......................................................................................................184

Figure 5-3. Effect of ASD (with/without biofungicides) and biofungicides on (A)

cumulative anaerobic condition, (B) soil pH, and (C) gravimetric soil moisture

content. ...................................................................................................................................185

Figure 5-4. Effect of ASD (with/without biofungicides) and biofungicides on

percentage sclerotial germination from sclerotia recovered from (A) 5-cm

examined on PDA, (B) 10-cm examined on AIA, and (C) 15-cm examined on

TSM. ......................................................................................................................................186

Figure 5-5.Effect of ASD (with/without biofungicides) and biofungicides on (A)

percentage sclerotial parasitism by Trichoderma, (B), zygomycetes, (C) other

fungi, and (D) bacteria examined on PDA. Sclerotia were recovered from a

depth of 5-cm. ........................................................................................................................187

xvi


percentage sclerotial parasitism by actinomycetes on AIA. Sclerotia were

recovered from a depth of 10-cm. ..........................................................................................188


percentage sclerotial parasitism by (A) Trichoderma (B), zygomycetes (C)

other fungi, and (D) bacteria on AIA. Sclerotia were recovered from a depth

of 10-cm. ................................................................................................................................189

Figure 5-8. Effect of ASD (with/without biofungicides) and biofungicides on (A)

percentage sclerotial parasitism by Trichoderma (B), zygomycetes (C) other

fungi and (D) bacteria examined on TSM. Sclerotia were recovered from a

depth of 15-cm. ......................................................................................................................190

Figure 5-9. Effect of ASD (with/without biofungicides) and biofungicides on soil

population of actinomycete spp. ............................................................................................191


soil populations of Bacillus spp. ............................................................................................192


soil populations of Trichoderma spp. ....................................................................................193


cowpea nodules number and mass. ........................................................................................194

Figure 5-13. Effect of amendment C:N ratio on percentage root colonization after

ASD treatment, 2014. ............................................................................................................195


root biomass of cowpea and tomato. ......................................................................................196


shoot biomass of cowpea and tomato. ...................................................................................197

Figure 5-16. Phylogenetic analysis of major soil microbial fungi identified in

ASD treated soil. Sequences were aligned using ClustalW and phylogenetic

tree was constructed using Mega 5.6. ....................................................................................198

xvii

ACRONYMS

AM Arbuscular mycorrhizal

ASD Anaerobic soil disinfestation

AIA Actinomycete isolation agar

BSD Biological soil disinfestation

BIA Bacillus isolation medium

C Carbon

C:N Carbon to nitrogen ratio

CFU Colony forming units

Fol Fusarium oxysporum f. sp. lycopersici

IRIS Indicator of reduction in soils

MeBr Methyl bromide

NH4 Ammonium

NO2 Nitrite

NO3 Nitrate

OA Organic amendment

ORP Oxidation-reduction electrodes

PDA Potato dextrose agar

TSM Trichoderma selective medium

Sr Sclerotium rolfsii

VFA Volatile fatty acid

1

Introduction

Methyl bromide (MeBr) is a very effective pre-plant fumigant against soil pests. However, it is a

serious ozone depleting fumigant; therefore, its use was banned as part of an international treaty

known as the Montreal Protocol. Several chemical and non-chemical approaches have been

introduced as a replacement for this effective soil fumigant. Many chemical alternatives have

been registered, but these chemicals do not reach the level of broad spectrum control, with many

shortcomings on effectiveness, consistency and safety. Anaerobic soil disinfestation (ASD;

synonymous to biological soil disinfestation) appears to be a potential non-chemical alternative

to MeBr, which has been successfully implemented or researched in Japan, the Netherlands,

Florida, California (Blok et al., 2000; Butler et al., 2012b; Messiha et al., 2007; Momma et al.,

2013; Shennan et al., 2007), and other parts of the world (Figure A1). Other non-chemical

alternatives have shown promising results, including flooding, solarization, steam sterilization

and biofumigation but these alternatives are either effective only in certain regions or cost

prohibitive. For Tennessee vegetable crop production, ASD has shown some promise (McCarty

et al., 2012a) but more research and commercial development is needed to make ASD

technically and economically feasible for the state and the southeastern region.

ASD utilizes locally available organic amendments (OAs) as carbon (C) sources, and unlike

many chemical fumigants, is safe to use near residential areas without any safety concerns.

Therefore, it is potentially an economically and environmentally sustainable technique for soil

disinfestation. Further, ASD has shown efficacy against many soilborne pathogens such as

Fusarium oxysporum, Sclerotium rolfsii, Verticillium dahliae, Pythium ultimum, Rhizoctonia

solani, Ralstonia solanacearum, and others (Butler et al., 2012b, Shennan 2014 ). Quantitative

analysis of ASD studies on soilborne pests has not been reported and the meta-analytic review of

previously published results on pest suppression due to ASD is useful to understand the efficacy

of ASD practice.

In ASD, OAs provide labile C sources to soil microbes that create anaerobic conditions through

increased microbial activity in moist, plastic mulched soils (Butler et al., 2012a; Butler et al.,

2012b). The antagonistic properties and pesticidal compounds are generated in the soil by

indigenous anaerobic microorganisms with the production of volatile fatty acids, which act

2

against plant diseases and pests (Blok et al., 2000). The ratio of C:N, as well as the rate and

solubility of the C source, plays a critical role in microbial growth and metabolism (Nicolardot et

al., 2001), plant growth and crop nutrition (Akhtar and Malik, 2000). Different OAs, such as

grasses, wheat bran, molasses, potato haulms, cruciferous plants and cover crops have been

examined as an ASD C source but no study has identified a suitable C:N ratio and carbon rates of

the ASD OAs. Recent studies in Japan with ethanol (Momma et al., 2013) and in Tennessee with

cover crops (McCarty, 2012a) as a C source amendment suggested on optimization of ASD for

its practical application. On the other hand it is also important to note that ASD has shown a

significant shift in the microbial community composition (Mazzola et al., 2012), and this is

considered due to application of organic amendments (Shennan et al., 2013). Bacillus spp.

(Momma et al., 2013) and Trichoderma spp. (Kredics et al., 2003) are well-known antibiotic and

toxin producers in controlling mechanisms of soilborne pathogens. Studies on various biocontrol

agents are in progress to replace chemical applications. Recently, actinomycetes were reported as

sclerotial parasites that have a positive response toward soil amendments. However, studies on

these microbes in response to ASD C sources are lacking and it is necessary to determine the

effect of ASD on these beneficial organisms.

This dissertation provides a meta-analytic review of ASD and consists of experiment to optimize

the ASD amendment C:N ratio and C rate to provide specialty crop growers with a pest control

tools for replacing MeBr and other chemical fumigants, while maintaining high value crop

production systems that are profitable and sustainable. This dissertation consists of five chapters.

The first chapter is a manuscript submitted to the journal, Frontiers in Plant Science,

summarizing the meta-analysis of efficacy of ASD on soilborne pathogens, nematodes, weeds,

and crop yield. The second chapter discusses a growth chamber study that examined the C:N

ratio of C amendments at 4 mg C g-1

of soil to optimize ASD to evaluate soil anaerobic

conditions and mortality of yellow nutsedge tuber. The third and fourth chapters summarize the

research conducted in growth chamber and field conditions to evaluate the effectiveness of dry

molasses and wheat bran maintained at different C:N ratios or C rates to suppress introduced

inocula of the Fusarium oxysporum f. sp. lycopersici and sclerotia of Sclerotium rolfsii

respectively. The fifth chapter is research study to evaluate the impact of ASD amendment

maintained at 4 mg C g-1

of soil on populations of Trichoderma, actinomycetes, Bacillus and root

3

colonizers. In this chapter, the impact of ASD amendment against germination and parasitism of

sclerotia of S. rolfsii is also examined.

4

Appendix

Figure A-1. Map of countries/states (in black) conducting ASD studies.

Washington

Argentina

China

United

Kingdom

Japan

The Netherlands

Sweden

California

Florida

Tennessee

North Carolina

5

Chapter 1

A meta-analysis of the impact of anaerobic soil disinfestation on pest

suppression and yield of horticultural crops

6

This chapter was originally published by Utsala Shrestha, Robert M. Augé, and David M. Butler.

Shrestha, U, R. M. Augé and D. M. Butler (2016 in press). A Meta-Analysis of the Impact of

Anaerobic Soil Disinfestation on Pest Suppression and Yield of Horticultural Crops. Frontiers in

Plant Science.

My primary contributions to this manuscript include data collection and analysis, results

interpretation and writing. Dr. Robert Augé contributed to the methodology and meta-analysis

section along with reviewing of figures and tables.

7

Abstract

Anaerobic soil disinfestation (ASD) is a proven but relatively new strategy to control soilborne

pests of horticultural crops through anaerobic decomposition of organic soil amendments. The

ASD technique has primarily been used to control soilborne pathogens; however, this technique

has also shown potential to control plant parasitic nematodes and weeds. ASD can utilize a broad

range of carbon (C) amendments and optimization may improve efficacy across environments. In

this context, a meta-analysis using a random-effects model was conducted to determine effect

sizes of the ASD effect on soilborne pathogens (533 studies), plant parasitic nematodes (91

studies), and weeds (88 studies) compared with unamended controls. Yield response to ASD was

evaluated (123 studies) compared to unamended and fumigated controls. We also examined

moderator variables for environmental conditions and amendments to explore the impact of these

moderators on ASD effectiveness on pests and yield. Across all pathogen types with the

exception of Sclerotinia spp., ASD studies show suppression of bacterial, oomycete, and fungal

pathogens (59 to 94%). Pathogen suppression was effective under all environmental conditions

(50 to 94%) and amendment types (53 to 97%), except when amendments were applied at rates

less than 0.3 kg m-2

. The ASD effect ranged from 15 to 56% for nematode suppression and 32 to

81% for weed suppression, but these differences were not significant. Significant nematode

moderators included study type, soil type, sampling depth, incubation period, and use of mixed

amendments. Weed suppression due to ASD showed significant heterogeneity for all

environmental conditions, confirming that these studies do not share a common effect size. Total

crop yield was not reduced by ASD when compared to a fumigant control and yield was

significantly higher (30%) compared to an unamended control, suggesting ASD as a feasible

option to maintain yield without chemical soil fumigants. We conclude ASD is effective against

soilborne pathogens and while not conclusive due to a limited number of studies, we expect the

same for nematodes and weeds given observed effect sizes. Findings should assist researchers in

exploring ASD efficacy in particular environmental conditions and allow for development of

standard treatment protocols.

Keywords: Anaerobic / biological soil disinfestation, meta-analysis, soilborne pathogens,

nematodes, weeds, suppression, yield

8

1. Introduction

Methyl Bromide (MeBr), a broad-spectrum soil fumigant, was completely phased out in 2005

(with the exception of critical use exemptions) due to its stratospheric ozone depleting nature.

Specialty crop growers have used this fumigant to control soilborne pathogens, nematodes, and

weeds since the mid-twentieth century. Due to restriction on its use, growers are seeking

alternatives that will provide comparable crop yield to that of MeBr. A number of chemical

fumigant alternatives have been registered as replacements to MeBr fumigation (Rosskopf et al.,

2014), but growers may not be willing or able to adopt them due to geographic limitations,

reduced efficacy, safety issues, and regulatory constraints of these chemicals (Csinos et al., 2002;

Martin, 2003). Further, worldwide awareness of environmental degradation and reduced-

pesticide agricultural concepts (Carvalho, 2006) is driving many growers to seek non-chemical

techniques to control crop pests. Non-chemical techniques such as flooding, solarization,

steaming, and biofumigation (with cruciferous plant residues) are some available options for

disease suppression. However, these generally environmentally friendly approaches have

limitations (Shennan et al., 2010; Muramoto et al., 2014), such as high use of water (Runia and

Molendijk, 2010; Runia et. al., 2014a), high temperature requirements (Katan, 1981), use of

costly equipment (Backstrom, 2002; Runia and Molendijk, 2010), and site-specific variability

(Larkin and Griffin, 2007; Lopez-Aranda, 2014), respectively.

Another promising non-chemical option available to growers is anaerobic soil disinfestation

(ASD), also known as biological soil disinfestation or anaerobically-mediated biological soil

disinfestation, has been studied since 2000 in Japan (Shinmura, 2004; Momma, 2008), the

Netherlands (Blok et al., 2000; Messiha et al., 2007) and the USA (Butler et al., 2012b; Rosskopf

et al., 2014; Shennan et al., 2014). This technique relies on organic amendments to supply labile

C to soil microbes to create anaerobic conditions in moist and plastic-covered soil. Soil microbes

consume available oxygen and depletion of oxygen shifts the balance toward facultative

anaerobes. Gases (such as CO2, NH3, H2S, CH4, and N2O) and volatile fatty acids (VFAs)

produced due to microbial decomposition of labile C during ASD lead to suppression of plant

pathogens and nematodes. Among these compounds, VFAs (e.g., butyric acid and acetic acid),

are particularly known to contribute to the soil disinfestation process (Momma et al., 2006).

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ASD is an environmentally friendly pest control practice (Porter et al., 2010; Shennan et al.,

2014; Rosskopf et al., 2015) where soil microbial growth can be enhanced, and soil fertility

potentially enhanced by the addition of organic amendments. A number of active research

programs across the world continue to refine ASD techniques to control plant pathogens,

nematodes, and weeds, and to further elucidate mechanisms of ASD treatment success (Shennan

et al., 2014). Although ASD incurs relatively low implementation costs when locally available

amendments are utilized, currently, ASD application in the USA has largely been limited to a

few organic crop producers and early-adopter conventional growers. ASD requires further

refinement of protocols to system variables and cost benefit analysis in comparison to other

chemical fumigants (Butler et al., 2012b; Shennan et al., 2014). Quantitative review of ASD

literature may be useful to researchers in terms of clarifying its efficacy across environments and

help to make more exacting recommendations for wide-scale adoption.

Only narrative reviews of ASD amendments and ASD comparisons in different countries have

been published (Shennan et al., 2014; Rosskopf et al., 2015; Strauss and Kluepfel, 2015).

However, a quantitative synthesis of the literature in reference to the efficacy of ASD on a range

of soilborne pathogens, nematodes, and weeds has not been reported. Meta-analysis is a powerful

tool that uses a set of statistical techniques to analyze independent studies quantitatively rather

than qualitatively (Ojiambo and Scherm, 2006). The meta-analytic approach has provided useful

results in medicine and psychology, and has been increasingly applied in agro-ecological

systems and pest management (Madden and Paul, 2011; Ngugi et al., 2011; Poeplau and Don,

2015). The purpose of this meta-analytic review of previously published results on pest

suppression due to ASD is to understand the efficacy of this non-chemical practice on a range of

soilborne pathogens, nematodes, and weeds. The meta-analysis also addresses comparative data

on pathogens, nematodes, and weeds using different moderator groups or explanatory variables.

Likewise, ASD effectiveness on crop yield is an important study group for meta-analysis that can

help growers make ASD adoption decisions. Many researchers rely on results from lab tests or

pot (e.g., greenhouse, growth chamber) studies only. However, soil disinfestation using organic

amendments under field conditions is a challenge for researchers as pathogen suppression is

subject to numerous environmental factors such as soil temperature, soil type, pathogen type, and

more (Bonanomi et al., 2010). Moderator analysis is thus important to understand how these

factors influence the efficacy of treatment. In this study, we examined the overall impact of

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various environmental and ASD treatment factors as moderators on ASD efficacy and effect size

of pest suppression and yield.

2. Materials and Methods

2.1 Data Collection

Literature databases were explored using the search engine Thompson Reuters Web of Science

on 20 August 2015. The terms used for the initial search, “soil disinfestation” OR “soil amend*”

OR “soil treat*”, returned 78,019 search results. These search results were filtered to 116 articles

using the search terms “anaerobic soil disinfestation” OR “biological soil disinfestation” OR

“reductive soil sterilization” OR “non-chemical fumig*” OR “non-chemical alternative*”.

Records were retrieved from Web of Science Core Collection (70), CABI (37), BIOSIS Citation

Index (6), and MEDLINE (3). Five books were excluded from 116 articles, and of the remaining

111 eligible articles, 65 were excluded because data described was presented in other original

articles, full text could not be found, or did not meet one of the following inclusion criteria

related to ASD-treatment: ASD treatment not applied, ASD was not compared with non-

amended control, or experiment was conducted in petri dishes only (Figure 1-1). In addition to

the remaining 46 articles, we identified nine additional eligible articles using ‘Google scholar™’

search. The meta-analysis included a total of 55 published and unpublished works (posters,

theses, and conference papers) spanning 16 years from 2000 to 2015 and written in English (50),

Japanese (2), Dutch (3) and Chinese languages (1).

We collected treatment means and sample sizes from each study to evaluate effectiveness of

ASD for pest suppression (soilborne pathogens/diseases, nematodes and weeds) and crop yield in

relation to 11 factors identified as moderator variables. If the means were reported in graphical

form, we used WebPlotDigitizer (Rohatgi, 2011) to estimate their values. ASD treatment means

were those that used any type of C amendment(s), soil saturation, or flooding and covering of

soil (usually polyethylene mulch) during the study period, while the non-amended and covered

or non-covered treatments were considered control means. Only for yield response, we also

collected means of fumigated treatments to compare with ASD treatment means. Multiple

treatments or pathogens from one article were treated as independent studies (sometimes referred

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to as paired observations in the meta-analysis literature) and represented individual units in the

meta-analyses. For example, Butler et al., (2012b) reported pathogen data for two trials for seven

different C amendments, resulting in 14 studies from that article. Although designating multiple

studies from one publication has the disadvantage of increasing the dependence among studies

that for the purposes of meta-analysis are assumed to be independent (Gurevitch and Hedges,

1999), the greater number of studies increases statistical power (Lajeunesse and Forbes, 2003).

This approach has been used commonly in plant biology meta-analyses (e.g. Holmgren et al.,

2012; Veresoglou et al., 2012; Mayerhofer et al., 2013; Chandrasekaran et al., 2014). The entire

data set included 900 studies from eight countries (Table 1-1).

2.2 Moderator variables

Several variables affecting pest suppression and yield were categorized and employed in

moderator analysis. Our first moderator of interest was the method of characterizing ASD

efficacy against each pest (i.e.,‘measure of efficacy moderator’), which represented studies that

reported ASD effectiveness against pathogen, nematode and weed abundance in various

quantifiable units (e.g., counts of pests, germination of pest propagules, ratings of disease; Table

1-2A). The different levels of ‘measure of efficacy’ were analyzed separately for each pest to

understand the variation in effect sizes (Figure 1-3). We categorized soilborne pathogens into 3

levels: bacterial, fungal or oomycete and within each are specific pathogens (Table 1-2B). We

also separated the beneficial soil organism Trichoderma to evaluate ASD effects. Further,

realizing importance of the Fusarium genus that has been widely studied, we categorized

Fusarium (F) spp. into 6 levels according to species and forma speciales (f. sp.) [Fusarium spp.,

F. oxysporum (F. o.), F. o. f. sp. asparagi, F. o. f. sp. cubense, F. o. f. sp. spinaciae, F. o. f. sp.

lycopersici]. We also categorized available studies on nematodes and weeds according to their

genus. Yield had two levels, non-amended control and fumigated control. We did not examine

total vs. marketable yield as a moderator due to insufficient studies representing the total

moderator level and we included total yield as a proxy for marketable yield where marketable

yield was not reported. In addition, we recorded information for six categorical environmental

moderators (explanatory variables) as study type, soil temperature, soil type, control type (with

or without plastic mulch), depth of sampling, and incubation period for both pests and yield

(summarized in Table 1-2F). These moderators are likely important determinants of the

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effectiveness of ASD in response to pest control and crop yield. In addition, ASD heavily relies

on amendments for C supplement and directly affect the ability of ASD to suppress pests.

Accordingly, ASD amendment was categorized in four moderators: form (liquid or solid), single

amendment or mixed, type, and rate (Table 1-2G). For environmental condition and amendment

groups all moderator levels may or may not be present in the analysis.

2.3 Effect size and meta-analysis

Our analyses followed the methodology and terminology of Borenstein et al. (2009) and were

guided by the criteria suggested by Koricheva and Gurevitch (2014). We computed summary

effects and associated statistics using Comprehensive Meta-Analysis Version 3 (CMA) software

(Biostat, Englewood, NJ, USA; 2014). We used a random-effects model for the meta-analyses,

considering that true effects are likely to have varied across studies (rather than a fixed-model,

which assumes the same value or true effect for all studies).

The effect sizes were calculated as the natural log response ratio (lnR) of treatment mean to

control mean and subjected to analysis of overall effect sizes (pest suppression and yield

responses) of ASD for each moderator. lnR for each observation was calculated as

lnR=ln(Xt/Xc)

where Xt is the ASD treatment mean and Xc is the control mean (non-amended, untreated or

fumigated control mean for yield). The log transformation was needed to balance positive and

negative treatment effects and to maintain symmetry in the analysis (Borenstein et al., 2009).

Given that approximately 80% of papers did not report a measure of dispersion, non-parametric

variance was calculated as:

VlnR=(nt+nc)/(nt∗nc)

where, VlnR is the variance of the natural log of the response ratio, and nt and nc are the samples

sizes of the treatment and control means, respectively. In studies in which several treatments

were compared with one control group, sample size of the one control group was partitioned

across treatment means. For example, for a study with one control and three treatments, each

http://journal.frontiersin.org/Journal/10.3389/fpls.2014.00562/full#B12

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having four replicates, the control sample size (4) was divided by three. This was done to avoid

overweighting studies by incorporating the same experimental units (e.g., plot, plants) in more

than one effect size. Values of zero are biologically common but mathematically not possible to

incorporate into meta-analysis (ratio denominator cannot be zero; cannot calculate the natural log

of 0). A common technique used in the medical literature is to add a small fixed number to any

zero value (NCSS, 2015). In pathogen control research, however, this technique yields very

inconsistent results, owing to the wide variety of units and the wide range of maximal pathogen

growth/survival values. Further, small non-zero values result in unreasonably inflated response

ratios. In order to analyze effect sizes of zero and near zero, we calculated 1% of the highest

pathogen abundance value for a study and raised any other value below 1% to that level: for

example, to 0.75 for 75 log CFU g-1

of soil, and to 0.03 for 3.0-cm colony diameter. Negative

values of pathogen abundance were equated to zero before applying the 1% adjustment.

Heterogeneity was assessed with the Q statistic, a measure of weighted squared deviations. Total

heterogeneity (𝑄𝑡) is composed of expected or within-study variation (𝑄𝑤) and excess or

between-study variation (𝑄𝑏). Heterogeneity was quantified using I2, a descriptive index that

estimates the ratio of true variation (heterogeneity) to total variation across studies:

I2= (𝑄𝑡 − 𝑑𝑓) 𝑄𝑡⁄ *100%

where 𝑑𝑓denotes the expected variation 𝑄𝑤 and 𝑄𝑡 − 𝑑𝑓 the excess variation (𝑄𝑏) (I2 is set to

zerowhen df exceeds 𝑄𝑡). A value of 0% indicates no true heterogeneity, and positive values

indicate true heterogeneity in the data set with larger values reflecting a larger proportion of the

observed variation due to true heterogeneity among studies. Assumptions of homogeneity were

considered invalid when p values for the Q test (Phetero) for heterogeneity were less than 0.1 (e.g.,

Bristow et al., 2013; Iacovelli et al., 2014). We assumed a common among-study variance across

moderator subgroups.

2.4 Publication bias and sensitivity analysis

Publication bias is the term applied to a body of research in the refereed literature that is

systematically unrepresentative of all completed studies (Rothstein et al., 2006). Literature

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reviews can be subject to publication bias, and the standard narrative review more so than

quantitative meta-analysis review (Borenstein et al., 2009). The issue is raised more often with

meta-analysis, likely because this method is intended to be comprehensive. The concern is the

possibility that significant treatment differences are more likely to be published than non-

significant findings. Direct evidence of publication bias is difficult to obtain, but it is important

to check for it (Sutton, 2005; Madden and Paul, 2011; Koricheva and Gurevitch, 2014). Methods

generally involve exploring the relationship between study effect size and precision. The idea is

that studies with smaller sample sizes or higher variance will tend to have larger effect sizes than

larger studies with greater precision. Hence, potential publication bias was assessed statistically

with Begg and Mazumbar rank (Kendall) correlation and represented graphically with funnel

plots of effect sizes versus their standard errors (estimated from their non-parametric variances)

(Begg and Mazumdar, 1994; Borenstein, 2005; Borenstein and Cooper, 2009; Borenstein et al.,

2009). The Duval and Tweedie iterative trim and fill method was used to demonstrate how the

summary effect size would shift if apparent bias were to be removed (Duval and Tweedie, 2000).

Sensitivity analysis was performed for the overall summary effects by removing one study and

re-running the meta-analysis for every study in the analysis. This shows how much each study

contributed to the summary effect, by noting how much the summary effect changes in its

absence. Possible temporal changes in effect size were evaluated with meta-regression using

publication year as a quantitative moderator (Koricheva & Gurevitch, 2014). Meta-regression

analysis was conducted with the CMA software, with the restricted maximum likelihood and

Knapp-Hartung methods (IntHout et al., 2014).

3. Results

We did not see evidence of publication bias. Visually, the funnel plots for each of the summary

effects showed no pattern that would reflect bias toward not reporting small positive or negative

effect sizes (Table 1-3). Large and small studies across the range of standard errors had the

expected variability around the summary effect size. Within the Begg and Mazumdar (1994)

rank correlation test, each of the summary effects had absolute Kendall tau values below 0.02,

indicating no publication bias (no tendency for effect sizes to increase as study size decreases)

(Table 1-3). The Duval and Tweedie (2000) trim and fill procedure imputes missing studies

15

needed to make the funnel plot symmetrical, removing the most extreme small studies and re-

computing the effect size at each iteration until the funnel plot is symmetric on either side of the

new (adjusted) summary effect. To maintain proper variance, the original studies are added back

into the analysis along with a mirror image for each. The adjusted value is suggestive only, as

when between-study heterogeneity exists (as was the case in our meta-analysis), trim and fill

may inappropriately adjust for publication bias, where none exists, and thereby led to spurious

changes in the summary effect (e.g., Terrin et al., 2003). A main concern about missing studies is

that their absence in the analysis may result in an exaggerated summary effect. In our analysis,

however, the summary value adjusted for potential missing studies is further from zero than the

original value for the pathogen and weed summary effects (Table 1-3). The test revealed no

potential missing studies and hence no adjustments for nematode control or yield assessed

relative to non-amended controls or to fumigated controls. Therefore, the trim and fill analysis

indicates no concern that publication bias has resulted in inflated summary effects. In fact, if the

suggested adjustments are legitimate for pathogen and weed control (if there really are missing

studies) then the Duval and Tweedie analysis points to an even greater impact of ASD in

controlling these pests.

The stability of the overall summary effects was assessed with sensitivity analysis. One study

was removed and the summary effect recalculated. This was repeated for all studies to determine

how much any one study affected the summary effect size. The study with the largest influence

on pathogen control was study 379 (lnR = -5.510, Verticillium treatment, Runia et. al., 2014b),

whose removal changed the summary effect by 0.4% (from a 67.5 to 67.1% reduction in

pathogens). The study with the largest influence on nematode control was study 720 (lnR = -

0.401, sandy soil with solid amendment treatment, van Overbeek et al. (2014), whose removal

changed the summary effect by 3.4% (from a 36.4 to a 33.0% reduction). The study with the

largest influence on weed control was study 794 (lnR = -0.810, trial 5, McCarty et al., 2013),

whose removal increased the size of the summary effect by 2.4% (from a 52.7 to a 55.1%

reduction in weeds). The study with the largest influence on yield was study 871 (lnR = 0.205,

eggplant treatment, (Butler et al., 2012b), whose removal reduced the summary effect by 5.9%

(from a 28.6 to a 22.7% promotion of yield, relative to unamended controls). Koricheva and

Gurevitch (2014) recommended testing whether a summary effect has changed over time, when

16

studies comprising the effect have been published over many years. Changes in the summary

effect could potentially result from publication bias, changes in methodology, or real biological

changes. Investigating chronology (year of publication), as a quantitative moderator using meta-

regression, ASD control of pathogens has changed slightly over time; the yearly average change

was -1.0% (p=0.81) over the data’s 16 publication years. ASD control of nematodes has changed

somewhat more over its 12 years of data, with an average decline of -1.8% per year (p=0.07).

There was an insufficient range of publication years of articles and studies to characterize the

influence of ASD on weed control or yield.

For our analysis, a natural log response ratio (lnR) value below zero indicates suppression of

pests (i.e., soilborne pathogens, plant parasitic nematodes and weeds), a value above zero

indicates an increase in pests with ASD, and a zero value signifies no effect of ASD treatments

on pest suppression. The levels within moderators are considered significantly different from

each other or from the overall mean when confidence intervals do not overlap. I2 and Phetero

characterized heterogeneity (the presence of underlying structure, i.e., true differences among

studies) within moderators. For each pest, we grouped our results as each pest type or crop type,

experimental condition and amendment used in ASD. We reported ASD yield response

separately for the fumigated control and unamended control.

3.1 Measure of efficacy

We detected an overall negative ASD effect on pathogen abundance in various quantifiable units

(-1.18 [CI -1.56 to -0.80]). When growth of pathogens was measured in colony size, ASD effect

was highest with 91% suppression and was significantly different from other units (Figure 1-2A).

Such a high significance in colony size was reported as pathogen suppression indicator during

ASD treatment in one article (Mazzola and Hewavitharana, 2014) with 15 studies, but realizing

the importance of the study and the slight difference in the overall effect size (5%) after removal

of the colony size unit, we decided to include all studies in our analysis. In the case of

nematodes, all units ranged between 20 to 40% and we observed 37% overall effectiveness for

nematode suppression (Figure 1-2B). Number of weeds in terms of ‘count’ (i.e., population or

density) was highly reduced by ASD compared to germination of weed propagules (82% vs.

29%; Figure 1-2C).

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3.2 Pathogens

Overall ASD effect on suppression of different soilborne pathogens, which were categorized as

bacterial, oomycete or fungal pathogens was -1.22 [CI -1.57 to -0.87] showing 70% suppression

over 533 studies (Figure 1-3). Suppression was significantly higher for oomycete pathogens than

for fungal pathogens and similar for oomycete and bacterial pathogens (Figure 1-3A). Between

oomycetes, Phytophthora had higher suppression by ASD than Pythium but the difference was

not statistically significant as CIs for the two summary effects overlapped (Figure 1-3B). More

studies on ASD were conducted for fungal pathogens (7 soilborne genera), among which

Sclerotinia was least suppressed by ASD (15%). ASD effect on Sclerotinia suppression

significantly differed from Fusarium suppression (70%). All soilborne pathogens except

Sclerotium were better suppressed by ASD (>63%) than non-amended controls although these

pathogens did not differ significantly (Figure 1-3C). Cylindrocarpon was the most suppressed

pathogen (86%), but with high CI values. To get an idea of the ASD effect on beneficial

organisms, we also evaluated ASD effect on Trichoderma (n=24) and we observed a positive

effect of ASD on these beneficial fungi (Figure 1-3D).

Since Fusarium was the most studied pathogen with 237 individual studies, it was of interest to

observe the ASD effect on different host specific Fusarium pathogens (f. sp.) within Fusarium

level. It also included uncharacterized F. oxysporum (54) and uncharacterized Fusarium spp.

(19). Overall effect size of Fusarium level within pathogen was -1.05, [CI -1.55, -0.54]

(representing an ASD suppression of 65% in raw terms), with significant heterogeneity p<0.001.

True variation among studies, estimated by I2, accounted for 13% of total variation. We observed

a significantly higher suppression level of ASD for the spinach and tomato wilt pathogens; F. o.

f. sp. spinaciae (87%) and lycopersici (74%), respectively. The uncharacterized F. o. also

showed a similar effect size and was significantly higher than other levels of Fusarium (76%).

The F. o. f. sp. cubense and other uncharacterized Fusarium spp. were less suppressed by ASD

(Figure 1-3E). When we compared the ASD effect on sclerotial germination percentage of

sclerotia-bearing pathogens, we found germination percentage was effectively lowered in

Verticillium, Rhizoctonia and Sclerotium, but not in Sclerotinia (Figure 1-3F).

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3.2.1 Experimental conditions for pathogen studies

Experimental conditions for pathogens included meta-analysis results from only soilborne

pathogens and excluding beneficial mycoparasites and non-amended treatments (e.g., flooding

only). Small studies carried out in the laboratory and growth chamber conditions showed 61%

pathogen suppression and large studies conducted in the field and the greenhouse showed

slightly higher suppression (72%, Figure 1-4A). At high soil temperature, the pathogen reduction

by ASD effect was ~10% higher than at moderate and lower soil temperatures (Figure 1-4B),

however, a significance difference was not observed due to extended confidence interval of high

temperature. The ASD treatment in volcanic soil from Japan showed significantly higher

suppression of pathogens than sandy soil (83%). While both types of soil did not differ with clay,

gray low land and loam soil. ASD effectiveness was significantly higher for ‘other media,’ which

included greenhouse media, perlites, etc. (94%; Figure 1-4C). Pathogen suppression was not

affected by whether ASD treatments involved covering (Figure 1-4D), and degree of suppression

has been similar across different sampling depths (64 to 71%; Figure 1-4E). ASD incubation

periods of greater than 10 weeks and 3 to 5 weeks were less effective than other periods. It is

interesting to see >78% pathogen suppression for the less than a three-week period. Three weeks

is by far the most used ASD incubation period for pathogen suppression (222 studies) and is

among the most effective periods (64%; Figure 1-4F).

3.2.2 Amendment effect on pathogen suppression

The type and amount of amendment is a crucial component of ASD to provide labile C to

microbes, and so we examined amendment characteristics for influence on the efficacy of ASD

on pathogen suppression. Across all pathogen studies (n=533), five amendment moderators were

categorized and analyzed separately. Figure 1-5A provides results of liquid vs solid amendments

(n=533) and Figure 1-5B depicts mixed vs non-mixed amendments (n=533). We found 533

studies were amended with various C sources (Figure 1-5C) and 41 studies were non-amended

and were analyzed separately (Figure 1-5E). Ethanol, organic acid and other C source (glucose,

sucrose and xylose) in amendment type moderator are applied as liquid amendments. Besides

liquid molasses included in ag-by-product, all other amendments were solid amendments.

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Amendment in liquid form was more effective than solid form, 77% vs 64% (Figure 1-5A).

Mixing different amendment types did not increase the effectiveness of ASD as compared to

single amendment (Figure 1-5B). Most C amendments significantly reduced pathogen measures

(Figure 1-5C) and overall ASD effect on plant pathogens was -1.24, (CI [-1.56, -0.91] p<0.001).

When ASD was conducted with ethanol, ASD effectiveness increased dramatically and was

significantly different from other amendments: organic acid, combination, ag-by-product,

cruciferous, grass, and legume (91%). ‘Other C source’, which includes glucose, sucrose and

xylose showed the most pathogen suppression among amendments. Suppression of pathogens

was however lower than 61% when amendments were cruciferous, legume and grass. We also

examined anaerobic and flooding situations (i.e., without C amendment) to gain a sense of

whether pathogen survival under these conditions was similar to ASD treatment and we found

that while flooding was effective, anaerobic conditions are not as effective as ASD (28%, Figure

1-5D). Effectiveness of ASD on pathogen suppression also relies on rate of amendments.

Amendment rates less than 0.3 kg m-2

and 5 to 6 kg m-2

did not show as much suppression as

other rates (Figure 1-5E). Generally, the trend was that higher suppression was observed with

higher rates of amendment but in meta-analysis of amendment rate, we could see response of

pathogen suppression is not only subject to application rate.

3.3 Nematode suppression

Over all studies, ASD decreased nematode abundance by 37% (lnR = -0.45), with the confidence

interval slightly overlapping zero (p = 0.066; Figure 1-2B). The four individual efficacy

measures ranged from 20 to 40%, with confidence intervals also crossing zero. Among the three

most studied plant parasitic genera, ASD-induced inhibition was significant only for Globodera,

at 56% (Figure 1-6A). The summary effect was not significant for Pratylenchus, Meloidogyne

and the 3 genera grouped as ‘Other’. Among the six moderators characterizing experimental

conditions, most have at least one level with a significant ASD effect (Figure 1-6B-G). Unlike

pathogen suppression, ASD has resulted in substantial nematode suppression in large studies

(63%, p=0.002), with no suppression in small studies (38%, p=0.40) (Figure 1-6B). Suppression

was greatest at moderate soil temperatures (68%, p=0.01) and insignificant at the higher and

lower reported temperatures (Figure 1-6C). The ASD effect varied with soil type, with

significant suppression of nematodes (94%) occurring only in loam soils (Figure 1-6D). The size

20

of the ASD-induced suppression has not differed as a function of its comparison to uncovered vs.

covered controls (Figure 1-6E). Sampling depth markedly affected estimation of ASD efficacy,

with nematodes reduced by 82% and 70%, respectively, in deep and shallow regions of the soil

profile, while at moderate depth a near significant ASD stimulation of nematodes has been

observed (Figure 1-6F). Incubation of less than 2 weeks has dramatically promoted nematode

survival, while an incubation of 4 to 6 weeks has resulted in significant nematode suppression

(Figure 1-6G). Amendment characteristics have had less influence on the extent to which ASD

suppressed nematodes than fungal pathogens (Figure 1-6H, I, J, K). Liquid and solid forms of

amendment have given similar nematode control (Figure 1-6H). Not mixing amendments has

been far more efficacious than mixing them (Figure 1-6I). None of the amendment types resulted

in a significant effect of ASD on nematode suppression (Figure 1-6J), although the small

numbers of studies representing several of the amendment types give low statistical power for

resolving differences. It was surprising that ASD showed nematode suppression at amendment

rates less than 2 kg m-2

and 3 to 4 kg m-2

, but rates at 2 to 3 kg m-2

and 4 to 5 kg m-2

did not show

any significant effect (Figure 1-6K), but again, the relatively low number of studies which were

performed under varying amendment types and soil temperatures limits interpretation.

3.4 Weed suppression

Few studies have addressed the influence of ASD on weed suppression (88 studies from 5

publications) and all studies were conducted in sandy soil. Overall weed reduction was 63%

when examined as both weed count and germination percentage (Figure 1-2C). Weed measures

have been much more affected by ASD when assessed as weed population density (82%,

p<0.001) than as germination of introduced propagules (29%, p=0.189). Chenopodium album,

Cyperus esculentus (yellow nutsedge), and less frequently studied species have shown significant

reductions with ASD (Figure 1-7A). Digitaria sanguinalis (crabgrass) has not been affected by

ASD in the few studies reported, and growth of Amaranthus retroflexus (pigweed) has actually

been substantially promoted by ASD. Large-scale application of ASD has resulted in significant

weed suppression whereas small-scale application has not suppressed weeds (Figure 1-7B). The

effect of ASD has been evident only when soil temperatures are high (Figure 1-7C). ASD

treatments have suppressed weeds only when compared to uncovered controls; covering soils has

given better weed control than ASD treatments (Figure 1-7D). Another interesting observation

21

for ASD was seen for sampling depth (or burial depth), with shallow depth being significantly

more suppressive to weeds and moderate depth promoting weed populations (Figure 1-7E).

Incubation periods of greater than 10 weeks showed far better control than a three-week period,

with the latter having little effect on weed measures (Figure 1-7F).

Each of the four amendment moderators affected ASD efficacy on weed suppression (Figure 1-

7G-J). The applied liquid form showed 97% weed suppression, about twice as effective as solid

amendments at 44% weed reduction (Figure 1-7G). Mixed and single amendment forms of ASD

have had similar, significant effects (Figure 1-7H). Among amendment types, ag by-products,

manure, ethanol and the less frequently used other C sources led to substantial ASD-induced

weed suppression in the range of 77 to 97% (Figure 1-7I). ASD resulted in significant weed

suppression only when the rate of amendment was greater than 1 kg m-2

(Figure 1-7J).

3.5 Yield

ASD treatment promoted yields of eggplant when compared to both non-amended and fumigated

controls (>130%, Figure 1-8A). Yield of bell pepper, strawberry, tomato, potato, and other crops

has remained unaffected by ASD. The lack of effect on yield occurs whether ASD efficacy is

viewed relative to non-amended or fumigated controls (Figure 1-8A-E). The absence of ASD

influence on yield has not been affected by study type (Figure 1-8B). ASD tended to promote

yield at sandy soil (33%, Figure 1-8C), higher temperatures (>54%, Figure 1-8D) and shorter

incubation times (34%, Figure 1-8E). Yield response increased to 6% when ASD was compared

with fumigated treatments and 30% with non-amended control (Figure 1-9A). ASD effect on

yield compared to both control treatments was highest for solid amendments compared to liquid

(15 to 32%, Figure 1-9B). Mixing of amendments increased yield 13 to 14% in both cases

(Figure 1-9C). Similar to weed suppression, manure amendment tended to have the most positive

effect on yield in both cases (>78%, Figure 1-9D). In addition, yield response increased with

respect to increase in application rate of amendment (Figure 1-9E).

22

4. Discussion

4.1 Is ASD effective for pathogen suppression?

Our results indicate strong evidence of pathogen suppression by ASD and that ASD plays a

critical role in minimizing pathogen inoculum by inhibiting germination of inoculum or reducing

the vigor of pathogens. We observed that colony size as a ‘measure of efficacy’ of pathogen

suppression was highly sensitive to ASD. Colony size during ASD would likely be affected by

the range of volatile compounds and other toxic anaerobic decomposition by-products. Along

with colony size, we also observed suppression of pathogens in terms of colony forming units,

germination percentage, infection percentage, and microsclerotia production. Given the various

efficacy measurements, we confirmed that overwintering forms of pathogens that impact crops

could potentially be effectively suppressed by ASD.

Studies have shown that ASD is effective in suppressing various soilborne pathogens (as

reviewed by Shennan et al., 2014; Rosskopf et al., 2015) and our meta-analysis results were

concurrent with those narrative reviews. Our meta-analysis also demonstrated the importance of

statistical power in terms of study number; for example, the only two studies for Cylindrocarpon

(infection percentage) showed no statistical difference, although disease reduction was 86%.

Banana wilt by F. o. f. sp. cubense was reported by Huang et al. (2015) and Wen et al. (2015) in

China and we are not surprised that these were less suppressed than all other Fusarium spp. as

treatments where flooding of soil and use of amendments like rice and corn straw, which have an

altered microbial response than that of more labile C amendments. We observed a significantly

higher suppression level of ASD for the spinach wilt pathogen F. o. f. sp. spinaciae (87%) and

tomato wilt pathogen Fo f. sp. lycopersici (76%). For Sclerotinia, which was less affected by

ASD, data was reported only from species sclerotiorum and it was reported that sclerotial

germination was highly influenced by the low amendment rate and soil temperature (Butler et al.,

2014b). Further, sclerotial viability, release of biochemical compounds, and infection ability vary

under different growing conditions and ineffectiveness of ASD in such cases may relate to a

combination of factors. At the same time, Thaning and Gerhardson (2001) reported sclerotia of

S. cepivorum from onion was unaffected by ASD (since data was not reported, Sclerotium

cepivorum is not included in the meta-analysis). On the other hand, sclerotia of Verticillium and

23

Sclerotinia both failed to survive in the same study. Variability in sclerotial infection

mechanisms (e.g., production of apothecia or mycelium; Imolehin et al., 1980) can also impact

ASD effectiveness. Nevertheless, from our meta-analysis, we can grasp the degree of fungistasis

(soil property preventing germination of viable propagules) being enhanced in ASD relative to

size of sclerotia; specifically, compared to Sclerotinia, smaller sclerotia of other sclerotial

pathogens are more effectively suppressed by ASD (see Figure 1-3F). Macrophomina, although

a sclerotia producer, the size of sclerotia are too small to enumerate (100um-200um) so sclerotial

germination is typically not reported. Recent studies on the bacterial pathogen Agrobacterium

tumefaciens in tree crops was reported to be suppressed by ASD (Strauss et al., 2014),

confirming ASD can be expanded to target other new plant pathogens and other crops.

Interestingly, our meta-analysis showed that ASD promoted the population of the mycoparasite

Trichoderma. This mycoparasite along with other fungi parasitizing sclerotia of S. rolfsii were

reported in Shrestha et al. (2013) and Thaning and Gerhardson (2001). Likewise, occurrence of

the S. sclerotiorum sclerotial parasite Coniothyrium minitans was reported by Thaning and

Gerhardson (2001). However, ASD effects on these beneficial organisms are not reported.

Looking at the positive impact of ASD on Trichoderma, although nonsignificant in this study,

suggests that more studies on ASD effects on beneficial microorganisms are needed. Studies

have revealed that Firmicutes, Clostridia, and Bacillus are prominent in microbial communities

during ASD (Mowlick et al., 2012). Further studies will help to further elucidate dynamics of

beneficial organisms during and post-ASD treatments, which will allow for treatment adaptations

to increase impact on beneficial organisms.

4.2 Conditions favoring ASD effectiveness on pathogen suppression

Our analysis suggests that ASD can work as a replacement to chemical fumigants for pathogen

suppression as we observed consistent pathogen suppression under various conditions (Figure 1-

4). These results also suggest that ASD significantly suppresses pathogens across a range of

temperatures. ASD treatments were more effective under higher soil temperature for both

nurseries and field conditions. If soil temperature is relatively high (>16°C ) the incubation

period can be reduced to less than 3 weeks since our analysis showed >80% of pathogen

suppression is achieved when temperature ranged from 16 to 30°C and pathogens were not

24

suppressed (40%) when temperature was low (data not shown). However, under low temperature

(<16°C ), ASD can be effective when certain factors are modified, for e.g. Ralstonia and

Verticillium under low temperature were effectively suppressed when higher amendment rates

(grass) and longer incubation periods of 10 to 25 weeks were practiced.

It is not uncommon to see greater suppression of pathogens in media such as potting soil and

other laboratory media other than soil, potentially due to reduced heterogeneity and reduced

populations of other soil microorganisms than in field conditions. These media based studies are

usually accompanied by smaller studies in a greenhouse, growth chamber, or laboratory with

controlled environmental conditions. Among various types of soil, clay and sandy soils showed

low suppression of pathogens in response to ASD treatment. Reasons for this observation may

include low availability of C to microorganisms due to rapid loss of soluble C in sandy soil and

greater adsorption and reduced water infiltration rate that affects the distribution of

decomposition by-products in clay soils. Clay soils are also likely to be more buffered against

changes in soil pH that may affect the accumulation of VFAs. Further, these acids are weakly

adsorbed to the soil’s exchange phase and have rapid turnover rate with short half-life (Jones et

al., 2003) and transitory when exposed from anaerobic to aerobic condition (Lazarovits et al.,

2005). Whereas volcanic ash, loam and gray lowland soil showed more suppression than clay

and sandy as these soils are themselves more fertile with high mineral contents, which often

enhance microbial activity.

One of the benefits of ASD is that it may be able to control pathogens under relatively short

incubation periods for a biological soil treatment. Surprisingly, ASD suppressed pathogens under

relatively short incubation periods. For an incubation period <3 weeks, we noticed 80% pathogen

control which was directly related to study type and soil type. Most of the studies less than 3

week incubation periods were reported from small-scale studies, including lab studies and other

C sources with eight amendment types in this analysis (110 studies) and only 24 studies reported

from large-scale studies, which included volcanic ash and gray lowland studies. Lower

percentages of pathogen suppression for 3 to 5 week and >10 week incubation periods may be

attributed to few amendment types (ag-by-product, brassica, grass or protein-by-product)

included in the meta-analysis. Pathogen suppression even after ASD treatment (post ASD)

25

duration reveals that ASD prevents resurgence of pathogens. However, post ASD treatments in

this analysis included only organic acid as the C amendment and this case may not be same for

the other amendments.

4.3 Contribution of ASD amendments to pathogen suppression

Amendments such as ethanol, organic acids, and liquid molasses are easier to apply in the soil

through drip application or by spraying. Liquid amendments are easily incorporated in soil and

rapidly translocate to the soil profile, which our results suggest makes them more effective in

ASD than solid amendments. In Japan, ethanol for ASD is already practiced at a relatively large

scale (Momma et al., 2013) and in Florida liquid molasses is commonly used (Butler et al.,

2012a; Rosskopf et al., 2014).

The categorization of amendment types in Figure 1-5C as moderator levels clearly shows

differences in various C amendments for pathogen suppression. It also indicates the importance

of moderator analysis as we get a clearer indication of effect sizes for various amendments. The

category ‘other C sources’ in this analysis (glucose, xylose and sucrose) showed the highest

suppression of pathogens, and studies were conducted in plastic boxes against Fusarium

pathogens. This illustrates that ASD is highly effective in controlled environments, likely due to

high anaerobic activity and confinement of VFAs and other volatile compounds (Hewavitharana

et al., 2014). Recently, Daugovish et al. (2015) used diluted glycerol as liquid amendment in

field soil and found that this C source was not as effective as rice bran to create long lasting

anaerobic conditions, which suggests that ASD effectiveness may in some cases differ in field

conditions.

From our analysis, ethanol is established as the most effective ASD amendment in controlling

plant pathogens. ASD effectiveness due to ethanol is directly related to concentration and

incubation period (Momma et al., 2006); a minimum incubation of 9 days is required for

effective ASD treatment when 0.5% (of soil volume) of ethanol is used. In addition, almost all

amendments used as C sources in the studies in this meta-analysis are considered to produce high

VFAs relative to non-amended controls (Figure 1-5).

26

For effective disease suppression, relatively high rates of amendment incorporation are reported

as necessary (Mowlick et al., 2013; Butler et al., 2014b). From our results, we confirmed higher

amendment rates lead to higher suppression. However, amendment rates at 5 to 6 kg m-2

rate

showed slightly less suppression and the reason may be that represented studies utilized only

grass and cruciferous plants. These amendments are less readily decomposed due to more

complex C compounds in whole plant tissue than in simpler and more labile C sources such as

ethanol, molasses, and glucose. Our results do suggest that ASD implementation costs could

potentially be lowered by application of low amendment rates in some cases (~ 300 g m-2

) of

amendment, which should be studied further.

4.5 ASD effect on nematode suppression

Measure of efficacy results indicated that hatching and number of nematodes, infection

incidence, and density of nematodes in roots were not significantly suppressed by ASD

treatment. Only potato cyst nematode (Globodera) was effectively controlled by ASD and half of

studies used protein-by-product amendment (Runia et al., 2014b; Streminska et al., 2014; van

Overbeek et al., 2014). Meloidogyne and Pratylenchus showed some suppression, but this was

non-significant. Nematode studies were approximately 7 times fewer than pathogen studies and

Figure 1-6 shows how this low number of studies affected nematode suppression evaluation,

with large confidence intervals due to error (Borenstein et al., 2009). We observed that nematode

suppression with ASD is not as effective as pathogen suppression. However, higher suppression

of nematodes by ASD treatments in field conditions, high organic content soil (e.g., loam and

volcanic soil) and 2 to 6 weeks of incubation period was observed. Both liquid and solid

amendments seem effective in nematode control. Besides manure and combination levels, all

other amendments were applied in ASD suppressed nematodes. In our analysis, moderator levels

manure and combination consist of poultry litter (7 studies each), which is known to have

nematicidal activity (Riegel and Noe, 2000) and was always associated with soil solarization to

increase soil temperatures. However, it was not effective enough for nematode suppression.

Since the early twentieth century, studies have revealed that decomposed organic matter helps in

reduction of nematodes (Linford et al., 1938). Reviews on various amendments and mechanisms

of suppression against various nematodes are reported (Rodriguez-Kabana, 1986; Rodriguez-

Kabana et al., 1987; Oka et al., 2007), but very few studies have been conducted to evaluate

27

efficacy of ASD on nematode suppression. More studies are encouraged under a range of ASD

treatment factors and environmental conditions in order to better evaluate ASD impact on plant

parasitic nematodes.

4.6 ASD effect on weed suppression

Although there are few reports on weed suppression by ASD compared to pathogens and

nematodes, our meta-analysis indicated that ASD is effective in suppressing weeds as well.

Except Amaranthus retroflexus, all other weeds evaluated were found to be suppressed with

ASD treatment (Figure 1-7B). Amaranthus is troublesome persistent weed with an extended

germination period (Karimmojeni et al., 2014) and the study included in our meta-analysis was a

pot observation thus emphasizing the need for additional research. Digitaria suppression likely

needs some refinement in ASD while Cyperus tuber germination was suppressed by ASD.

Although these weed suppression studies were conducted in pots, we believe that ASD can be

equally effective if used in field condition as Chenopodium album and other weeds in field study

showed high suppression when grass and other C sources were used as amendments. An ASD

effect on weeds at shallow depths with almost 100% control of weeds could potentially be of

large benefit; however, this represents few observations (n=9) which were reported from a single

paper (Muramato et al., 2008) conducted in pots, with high temperature and without a covered

control. More studies are needed with more variables for such cases to better assess suppression

effects. When amendments were in liquid form, almost 99% weed control was achieved and

reasons are likely similar to that discussed previously for pathogen suppression. It was not

surprising to see that ethanol and manure amendments in ASD are more capable of weed

suppression than other amendments as these may promote more toxicity than other C sources to

control the weed propagules. But, there is a need to explore more cover crops, ethanol and

manures as ASD amendments, and for an increase in the number of these studies. For effective

weed suppression, rates of amendments greater than 1 kg m-2

are likely needed.

4.7 ASD effect on crop yield

We found that total fruit yield of crops was not reduced by ASD when compared to a fumigant

control and yield was significantly higher when compared to a non-amended control. Our results

28

indicate that ASD is promising for sandy soil and high soil temperature and the result may be

due to suppression of pathogens and weeds by lethal temperatures, as well as substantial

beneficial effects of organic matter additions on chemical, biological, and physical properties of

sandy soils (Butler et al., 2014a). Application of manures and increased amendment rate

increased the yield (>50%) compared to both fumigated and non-amended controls. However,

due to low number of studies, we see overlapping of confidence intervals and it is expected that

if the number of studies on ASD using manures increases, we may see a significant crop yield

result from meta-analysis.

Not surprisingly, a far higher number of publications on ASD are related to disease suppression

than to yield response. The small numbers of published yield studies do not allow a

comprehensive meta-analysis. This, and the numerous variations inherent to field studies, led to

large CIs and likely insufficient power to determine with statistical confidence if yield summary

effects differ from zero. Further, analysis of yield data faces several limitations. First, many

papers do not report standard deviation and so use of non-parametric variance may have added

additional uncertainty to our results. Second, although our mean yields include mostly

marketable yield, in some instances (20 studies from McCarty et. al., 2014) we included total

yield as a proxy for marketable yield where marketable yield was not reported. As concluded by

(Belova et al., 2013), the lack of detail provided in many studies about field experimental

protocols, horticultural practices and field management history hinders conclusive analysis. The

wide confidence intervals for yield in our results likely reflect the fact that yield is affected by

many environmental factors, soil factors and other cultural practices.

5. Conclusion

Given that pests evaluated in ASD studies differ widely in biological characteristics, it is not

surprising that biologically-based ASD treatments may differentially impact survival and growth

of these organisms. ASD treatment showed high reduction in bacterial (Ralstonia), oomycete

(Pythium and Phytophthora) and fungal (except for Sclerotinia) pathogen inoculum. Among

fungal pathogens, ASD response to pathogen supression was high for Cylindrocarpon,

Fusarium, Macrophomina, Rhizoctonia, Sclerotium, and Verticillium. Among different host

specific F. oxysporum pathogens, F.o. f. sp. spinaciae and F.o. f. sp. lycopersici were

29

significantly suppressed by ASD. Under most environmental conditions (i.e., a range of study

types, soil temperature, soil types and incubation period), suppression of pathogen inoculum due

to ASD treatment ranged from 50 to 94%. While our results indicate that ASD is effective for a

suppression of a broad range of plant pathogens as compared to an non-amended control across a

range of amendment types, amendment rates (>0.3 kg m2), soil temperatures, soil types, and

treatment incubation periods, research and demonstration studies often report variable results

when compared to conventional soil fumigant controls. While this is not surprising given that

ASD treatment relies on a more complex biological process that is influenced by environmental

conditions and interactions with existing soil biology as compared to chemical fumigants, it does

suggest that further refinement to improve ASD techniques could lead to more consistent field

suppression compared to fumigants. Accordingly, ASD methods likely will need refinement

based on the pests of interest and environmental conditions in a given production system. Due to

a limited number of studies and variability in reported research, we cannot conclude that ASD is

consistently effective in suppressing nematode or weed pests, although suppression has been

achieved for some species under specific environmental and treatment conditions. Given broad-

based suppression of plant pathogens under ASD treatments, future research should focus on

further improving consistency of ASD treatment for soilborne plant pathogens to improve

competitiveness of this biologically-based technique with conventional soil fumigants.

6. Acknowledgements

We are grateful to Dr. Arnold Saxton for his assistance with statistical analysis and Heather

Toler for her assistance in CMA analysis. Research supported in part by funding from the

National Institute of Food and Agriculture, U.S. Department of Agriculture, under award number

2012-51102-20293 (Methyl Bromide Transitions Program).

30

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38

Appendix

Table 1-1. Number of studies by country and USA states and response variables examined

S.no. Country Soilborne

pathogens1

Nematodes Weeds Yield Non-

Amended Trichoderma

1 Argentina 2 - - - - -

2 Belgium - 2 - - - -

3 China 56 - - 1 4 -

4 United Kingdon 4 - - - - -

5 Japan 84 2 - - 7 -

6 Netherlands 117 54 20 4 19 -

7 Sweden 12 - - - 8 -

8 USA (California) 36 - 3 56 - -

9 USA (Florida) 111 28 25 32 - 24

10 USA (Tennessee) 91 - 40 30 - -

11 USA (Washington) 20 5 - - 3 -

Grand Total 533 91 88 123 41 24

1Does not include studies without organic amendment (i.e., anaerobic and flooding only studies; 41 studies)

39

Table 1-2. Levels and attributes within each categorical moderator variable tested for

significance of pest suppression and yield responses

Categorical

moderator variables Levels Attributes

A. Measure of efficacy

(3levels)

Pathogen Colony size, germination (%), infection (%),

colony forming units (log), microsclerotia count

Nematode Mass in root (g), hatching (%), counts, rating of

disease

Weed Count, germination (%)

B. Soilborne pathogen

genera (3 levels)

Bacterial (1) Ralstonia

Oomycete (2) Phytophthora, Pythium

Fungal (7) Cylindrocarpon, Fusarium, Macrophomina,

Rhizoctonia, Sclerotium, Sclerotinia, Verticillium

C. Nematodes (4 levels) Plant parasitic

Globodera, Pratylenchus, Meloidogyne and others

(Heterodera, Pratylenchus, Trichodorus,

Tylenchorhynchus)

D. Weeds (5 levels) Weed type

Amaranthus retroflexus, Chenopodium album,

Cyperus esculentus, Digitaria sanguinalis and

others

E. Yield (2 levels) Control Fumigated control, non-amended control

F. Environmental conditions

i. Study type

(2 levels)

Small scale Study mostly in controlled environment using

glass, bag, bucket, box, pot, growth chamber

Large scale Field / plots

ii. Soil temperature

(3 levels)

Low <16°C

Moderate 16 to 35°C

High >35°C

iii. Soil type

(6 levels)

Sandy Sandy, sandy peat, sandy loam, loamy sand, sandy

clay loam, glacial sand

Clay Clay, clay loam

Loam Loam, silty loam, marine loam

Gray lowland Poorly drained soil

Volcanic ash Andosol

Other media Greenhouse soil, peat, perlite and other

iv. Control

(2 levels)

Yes Plastic sealed to create anaerobic conditions

No Uncovered treatment

v. Depth of sampling (3

levels)

Shallow 0 to 5-cm

Moderate 6 to 15-cm

Deep >15-cm

vi. Incubation period Variable Ranged from <3 weeks to>10 weeks

40

Table 1-2. contd.

Categorical

moderator variables Levels Attributes

G. Amendments

i. Amendments form (2

levels)

Liquid Ethanol, organic acids, semi-solid molasses

Solid All other amendment types

ii. Amendments mixed (2

levels)

No Single amendment only

Yes 2 or>2 different amendments mixed

iii. Amendment type (11

levels)

Agricultural by-

product

Wheat bran, rice bran/straw, maize stalks/straw,

molasses (solid and liquid), grape pomace, onion

waste, potato residue

Cruciferous Arugula, broccoli, radish, mustard and other

mustard products

Combination >2 amendments used

Protein by-product “Herbie1”, volatiles from Herbie

Legume Cowpea, crimson clover, hairy vetch, sunn hemp,

alfalfa

Grass

Oats, cereal rye, perennial ryegrass, Italian

ryegrass, pearl millet, sorghum-sudangrass, wheat

and other grasses

Manure Poultry litter with or without solarization,

composted steer manure

Organic acid Acetic acid, butyric acid, lactic acid, ‘SPK’

Ethanol Ethanol, bio-ethanol (0.5%, 1%, 2%)

Other C source Glucose, sucrose, xylose, C media (other organic

material)

iv. Non-amended No amendments Anaerobic or flooding

v. Rate per m2 Variable Ranged from <0.3 kg to>9kg

1Proprietary blend of plant products, see Runia et al., 2014b for more information.

41

Table 1-3. Measures used in characterizing publication bias for each effect size (after Borenstein,

2005)

Effect

sizes Summary effect

1 Funnel

plot2

Kendall

tau3

Duval &

Tweedie

adjusted6

No.

impute

n

lnR

p

No. var.

Pathogen 533 -1.12 <0.001 0.005 No -0.07 -1.29 66

Nematode 91 -0.04 0.027 0.060 No -0.14 -0.04 0

Weed 88 -0.75 0.002 0.058 No -0.11 -1.49 17

Yield with

non-

amended

control

68 0.26 0.034 0.015 No 0.02 0.26 0

Yield with

fumigated

control

55 0.05 0.687 0.018 No -0.07 0.05 0

1Summary effect: n=number of studies, lnR = natural log of overall summary effect, p=

probability that summary effect ≠ 0, No. var. = number of different variance values of studies

comprising the summary effect

2Funnel plot appears asymmetrical

3Begg and Mazumdar Kendall rank correlation: tau = rank correlation coefficient (with

continuity correction)

4Duval and Tweedie trim and fill: adjusted summary effect after imputing missing studies using

an iterative trim and fill procedure, No. impute = number of studies imputed in the trim and fill

exercise.

42

Figure 1-1. Flow diagram showing the study selection procedure

43

Comparisons among levels of (A) Pathogen (-hexagon symbols), (B) Nematode (-diamond

symbols) and (C) Weed (-square symbols). For each level of moderator, values to the right of

the CI line with negative effective size are percent pest suppression and with positive effect size

are percent of promotion. Number of studies reporting data for each level of moderator is given

in brackets. The moderator level was significantly different from zero if p-value ≤ 0.05. Values

below panel titles to the left are I2 (percentage of heterogeneity due to true variation among

moderator levels) and Phetero (test of the null hypothesis, that all studies share a common effect

size when it is greater than 0.1) for each moderator. Open symbols denote levels of each

moderator (subgroups); closed symbols denote overall moderator summary effect.

Figure 1-2. Weighted summary effect sizes (lnR) and 95% confidence intervals (CIs) for ASD

moderator ‘measure of efficacy’ (various measures of pathogen growth and survival used in the

literature).

44

Comparisons among levels of (A) Bacterial pathogen, (B) Oomycete pathogen, (C) Fungal

pathogen, (D) Beneficial mycoparasite, (E) Fusarium all and (F) Sclerotial germination. For each

level of moderator, values to the right of the CI line with negative effective size are percent

pathogen suppression and with positive effect size are percent of promotion. Number of studies

reporting data for each level of moderator is given in parentheses. The moderator level was

significantly different from zero if p-value ≤ 0.05. Values below panel titles to the left are I2

(percentage of heterogeneity due to true variation among moderator levels) and Phetero (test of the

null hypothesis, that all studies share a common effect size when it is greater than 0.1) for each

moderator.

Figure 1-3. Weighted summary effect sizes (lnR) and 95% confidence intervals (CIs) for

ASD effect on suppression of pathogens and beneficial mycoparasites.

45

Comparisons among levels of (A) Study type, (B) Soil temperature, (C) Soil type, (D) Control,

(E) Sampling depth and (F) Incubation period. For each level of moderator, values to the right of

the CI line with negative effective size are percent pathogen suppression and with positive effect

size are percent of promotion. Number of studies reporting data for each level of moderator is

given in parentheses. The moderator level was significantly different from zero if p-value ≤ 0.05.

Values below panel titles to the left are I2 (percentage of heterogeneity due to true variation

among moderator levels) and Phetero (test of the null hypothesis, that all studies share a common

effect size when it is greater than 0.1) for each moderator.


ASD effect on pathogen suppression under various experimental conditions.

46

Comparisons among levels of (A) Forms, (B) Mixed, (C) Types, (D) Non-amended and (E) Rate

per m2. For each level of moderator, values to the right of the CI line with negative effective size

are percent pathogen suppression and with positive effect size are percent of promotion. Number

of studies reporting data for each level of moderator is given in parentheses. The moderator level

was significantly different from zero if p-value ≤ 0.05. Values below panel titles to the left are I2



moderator.


ASD amendment effect on pathogen suppression.

47

Comparisons among levels of (A) Plant parasitic nematode (Other = Heterodera, Trichodorus

and Tylenchorynchus), (B) Study type, (C) Soil temperature, (D) Soil type, (E) Control, (F)

Sampling depth, (G) Incubation period, (H) Forms, (I) Mixed, (J) Types and (K) Rate per m2.

For each level of moderators, values to the right of the CI line indicate percent changes induced

by ASD in raw terms: negative values represent suppression or reduction, positive values

represent promotion. Number of studies reporting data for each level of moderator is given in

parentheses. The moderator level was considered significantly different from zero if its p-value ≤

0.05. Values below panel titles to the left are I2 (percentage of heterogeneity due to true variation

among moderator levels) and Phetero (test of the null hypothesis, that all studies share a common

effect size when it is greater than 0.1) for each moderator.


ASD effect on nematode suppression.

48

Figure 1-6. contd.

49

Comparisons among levels of (A) Weed type, (B) Study type, (C) Soil temperature, (D) Control,

(E) Sampling depth, (F) Incubation period, (G) Forms, (H) Mixed, (I) Types and (J) Rate per m2.

For each level of moderator, values to the right of the CI line with negative effective size are

percent weed suppression and with positive effect size are percent of promotion. Number of

studies reporting data for each level of moderator is given in parentheses. The moderator level

was significantly different from zero if p-value ≤ 0.05. Values below panel titles to the left are I2



moderator.


ASD effect on weed suppression.

50

Figure 1-7. contd

51


effect on yield response.

Comparisons among levels of (A) Crop type, (B) Study type, (C) Soil type, (D) Soil temperature

and (E) Incubation period. For each level of moderator, values to the right of the CI line with

negative effective size are percent yield decrease and with positive effect size are percent of

yield increment. Number of studies reporting data for each level of moderator is given in

parentheses. The moderator level was significantly different from zero if p-value ≤ 0.05. Values

below panel titles to the left are I2 (percentage of heterogeneity due to true variation among

moderator levels) and Phetero (test of the null hypothesis, that all studies share a common effect

size when it is greater than 0.1) for each moderator. Closed symbols () denote ASD compared

with non-amended untreated control; open symbols denote () ASD compared with fumigated

control.

52


amendment effect on yield response.

Comparisons among levels of (A) Control, (B) Forms, (C) Mixed, (D) Types and (E) Rate per

m2. For each level of moderator, values to the right of the CI line with negative effective size are

percent yield decrease and with positive effect size are percent of yield increment. Number of

studies reporting data for each level of moderator is given in parentheses. The moderator level

was significantly different from zero if p-value ≤ 0.05. Values below panel titles are I2



moderator. Closed symbols () denote ASD compared with non-amended untreated control;

open symbols () denote ASD compared with fumigated control.

53

Chapter 2

Effect of anaerobic soil disinfestation amendment C:N ratio on

tuber germination, growth and reproduction of yellow nutsedge

(Cyperus esculentus) and organic acid production

54

A version of this chapter is a manuscript in preparation for Weed Science by Utsala Shrestha,

Erin N. Rosskopf and David M. Butler.

My primary contributions to this manuscript include experimental setup, data collection and

analysis, results interpretation and writing. Dr. Rosskopf contributed constructive feedback to the

article.

55

Abstract

Anaerobic soil disinfestation (ASD) is a cultural technique to manage key pests especially

soilborne pathogens in a specialty crops using organic amendments, saturating and covering the

soil for at least a three-week period. ASD can be economic to growers if weed propagules

survival can be decreased with suitable organic amendments. Replicated growth chamber and

greenhouse studies were conducted in spring 2013 using two organic amendments, dry molasses

and wheat bran adjusted to four C:N ratios 10:1, 20:1, 30:1 and 40:1 ( 4mg C g-1

of soil) with a

non-amended control to evaluate ASD treatment impact on germination and growth of

introduced yellow nutsedge tubers. There was no interaction of C amendments and C:N ratios

observed for cumulative anaerobic condition, pH, soil N, nutsedge growth and tuber production

except for soil P. Mean cumulative anaerobic condition recorded for all treated pots was 58%

higher than in the control. We did not see any difference between dry molasses and wheat bran

as ASD amendments for cumulative anaerobic condition, soil pH, soil total nitrogen and carbon

to nitrogen ratio. Among the C:N ratio treatments, C:N 10:1 showed significantly lowest soil pH

(6.12) and soil C:N ratio (10.3) but highest total soil inorganic nitrogen (59.8 mg N kg-1

soil) and

inorganic phosphorus (28 mg P kg-1

soil) than the control and other C:N treatments (except soil

P). Regarding buried nutsedge tubers, mean non-germinated tubers recovered from pots were

higher for wheat bran (86%) than dry molasses (66%) and production of tubers from germinated

tubers were 44% more in dry molasses than wheat bran. Nevertheless, lowest non-germinated

tubers (23%) and increased in tubers production (40-60%) were obtained from control pots.

Tubers buried at 15-cm depth produced a greater number of large tubers (79%) than these buried

at 5-cm depth at all treatments. The wheat bran amended treatment successfully reduced the

shoot and root dry biomass compared to the control while dry molasses enhanced the shoot

biomass production. Among C:N ratios, germinated tubers and the number of both large and

small-sized tubers were lowest for C:N ratios 10:1, however, shoot dry biomass increased.

Results from this study indicate that while weed eradication using ASD technique was not

observed, both wheat bran and dry molasses used as ASD amendments resulted in higher tuber

mortality with lowest new tuber production than the control. Wheat bran at a lower C:N ratio

significantly reduced tuber germination and growth, hence emphasizing the importance of

56

amendment properties along with soil properties and nutrients for nutsedge tuber growth and

production during ASD treatment.

Keywords: Anaerobic soil disinfestation, C:N ratio, dry molasses, organic acid, organic

amendments, tuber, wheat bran, yellow nutsedge

57

1. Introduction

Yellow nutsedge is a noxious weed that competes with crops for light, soil nutrients, soil

moisture (Volz, 1977;Keeley and Thullen, 1978;Keeley, 1987;Morales-Payan et al., 2003a), and

can adversely affect crop plants by producing allelopathic compounds (Drost and Doll,

1980;Westendorff et al., 2013) and by serving as a host of fungal plant pathogens and nematodes

(Miller and Dittmar, 2014). Yellow nutsedge can be a problem if rhizomes are present in the

field (Stoller et al., 1972; Stoller and Sweet, 1987; Schippers et al., 1993), but a major concern

with yellow nutsedge control is its ability to produce large numbers of tubers (Anderson, 1999).

In yellow nutsedge, tubers are overwintering structures that can remain viable in the soil under

extreme climatic situations (Bendixen and Nandihalli, 1987) and a single plant can produce

hundreds to thousands of tubers per square meter (Tumbleson and Kommedahl, 1961) in a single

season. Morales-Payan et al. (2003b) buried 25 tubers/m2 in a plastic mulched tomato field,

which produced 2150 tubers/m2 in a single season showing potential of a single tuber to produce

86 additional tubers. Although, the number of tubers produced per unit tuber buried for the same

time period decreased drastically when initial buried tuber density increased from 25 to 50 and

100 tubers per square meter, tuber production often results in high economic loss. For example,

interference by these tubers during early growth stages can cause 45-50% of marketable yield

loss in tomato and pepper (Morales-Payan et al., 2003b; Bangarwa et al., 2011). Further, tubers

production in tilled soil can increase by 3 folds as compared to no-tilled plots (Johnson et al.,

2007).

Historically, the broad-spectrum fumigant, methyl bromide in combination with chloropicrin was

effective in minimizing nutsedge interference but the phase-out of the use of this fumigant has

necessitated new strategies to control yellow nutsedge (Rosskopf et al., 2005). Alternative

fumigant approaches to control yellow nutsedge tuber production are available, such as soil-

applied herbicides (Banks, 1983), drip applied herbicides ( halosulfuron, imazosulfuron, and

trifloxysulfuron) (Dittmar et al., 2012a; Monday, 2014) and many other chemicals. However,

under plasticulture systems, herbicide application is inconsistent, requires higher doses and more

frequent application (Dittmar et al., 2012a; Dittmar et al., 2012b)

58

Herbicides such as naproamide and trifluralin were inconsistent and ineffective at controlling

nutsedge, while registered herbicides like fomesafen, s-metachlor, imazosulfurom failed to

achieve season-long control of nutsedge when used alone (Miller and Dittmar, 2014). These

herbicides also need pre-mulching and post-planting application, increasing costs and consuming

more time and labor. Devkota et al. (2013) reported high rates of allyl isothiocyanate (AITC) and

metam sodium reduced yellow nutsedge tuber density compared to MeBr-treated plots.

However, metam sodium and 1, 3-dichloropropene + choloropicrin in tomato trials failed to

improve crop yield (EPA 2011). Regarding soil fumigants, Gilreath et al. (2005) tested the

efficacy of metam sodium along with 1, 3-dichloropropene, chloropicrin, and a combination of

these fumigants, but these approaches also did not meet expectations for control. Along with

these fumigants, many other chemical fumigants pose limitations in terms of buffer zone

requirements and regulatory contraints (for example, the loss of registration of iodomethane).

Further, several studies have reported crop injury due to the application of these chemicals

(Dittmar et al., 2012a; Devkota et al., 2013) and are obviously inappropriate for organic or

chemical-free production systems.

Fewer options are available for specialty crops in organic production systems. Cultural controls

such as hand weeding, and fallow tillage often require more labor and limit the growing season,

respectively. Mechanical weeding may also spread rhizomes, increasing weed distribution

(Keeling et al., 1990). Solarization with or without organic materials is found to be an effective

cultural practice to control nutsedge but requires temperatures lethal to tuber survival (Chellemi

et al., 1997; Johnson et al., 2007). Organic mulches (Chen et al., 2013) and biological control

practices (for e.g. Bactra verutana , Dactylaria higginsii and Puccinia canaliculata) can also be

options, but the effects of these organisms on tuber growth have not been tested (Keeley et al.,

1970; Charudattan, 2000; Morales-Payan et al., 2005; Riemens et al., 2008; Shabana et al., 2010)

and still need in-depth studies for commercial production (Evans, 1995; Li et al., 2003).

In plasticulture systems, a biofumigation study showed 50% reduction in tuber density, but this

did not reduce nutsedge competition (Bangarwa et al., 2011; 2012). Anaerobic soil disinfestation

(ASD), which was initially designed to control soilborne pathogens (Blok et al., 2000; Shinmura,

2004; Shennan et al., 2014) has been also studied for effectiveness against weed propagules and

yellow nutsedge tubers (Messiha et al., 2007; Muramoto et al., 2008; Butler et al., 2012b;

59

McCarty, 2012; Rosskopf et al., 2014a). This practice involves the incorporation of an organic or

carbon (C) amendment to supply a labile C source to microbes in order to create anaerobic

conditions under plastic (polyethylene) - mulched soils. Organic acids (Momma et al., 2006) and

reduced forms of iron and manganese (Momma et al., 2011) have been reported as major control

mechanisms in ASD. In the case of yellow nutsedge tubers, different C amendments especially

cover crops with C rate ranging from 0.5-2.5 mg C g-1

soil and C:N ratios ranging from 14:1 to

42:1 have been shown to reduce germination percentages compared to untreated controls (Butler

et al., 2012a; McCarty, 2012). However, large variation in suppression of tuber germination (35

to 70%) indicates that the practice may need optimization with characterization of amendment

C:N ratios to control weed germination, growth and reproduction. The appropriate C:N ratio is

necessary for subsequent crop performance, not only under aerobic conditions (Rodriguez-

Kabana et al., 1987) but also under anaerobic conditions of ASD (Butler et al., 2014b).

Moreover, the C:N ratio is known to be important for shifts in microbial population structure and

decomposition of organic matter (Akhtar, 2000; Högberg et al., 2007; Wan et al., 2015).

Previous studies on ASD microbial community changes investigated bacterial populations

(Momma et al., 2010; Mowlick et al., 2012; Mowlick et al., 2013a; Hong et al., 2014; Mowlick

et al., 2014) and fungal communities (Mazzola et al., 2012; Runia et al., 2014; Streminska et al.,

2014) showing biocidal effects on pathogens. Beneficial mycoparasites (Trichoderma) with

antagonistic activity against pathogens have also been isolated (Shrestha et al., 2013; Rosskopf et

al., 2014b) showing potential biological control effects associated with ASD treatment.

However, it is unknown how microorganism response to C:N ratio could impact tuber

germination at different amendment rates, or as amendment types and rates are changed, thus

affecting size and structure of the soil microbiome (Wan et al., 2015). Assuming enhanced

microbial activity and organic acid production during ASD could impact nutsedge tuber

germination, various C:N ratios were examined for their effectiveness in decreasing nutsedge

tuber survival and reproduction. For this, a growth chamber study was conducted with dry

molasses and wheat bran as ASD C amendments at four C:N ratios to evaluate the impact on

tuber germination and growth of yellow nutsedge and organic acid production.

60


2.1 Experimental setup

Experiments were arranged in an environmentally-controlled growth chamber in spring 2013, in

which the temperature was maintained at 25°C (14 h day) and 15°C (10 h night) to simulate soil

temperature regimes that represent the relevant production regions during spring in Tennessee

(McCarty et al., 2014), as well as many other production regions where ASD is under

investigation (Shennan et al., 2014). Soil from the ‘Ap’ horizon from the University of

Tennessee’s Organic Crops Unit, Knoxville, TN (previously planted with maize) and fine sand

were collected and sieved (<10 mm) to remove organic debris. The soil type is characterized as a

Dewey silt loam (fine, kaolinitic, thermic, Typic Paleudult) and at collection had a total carbon

(0.94%) and pH of 6.8. Treatment factors included two types of soil C amendments; dry

molasses (molasses) or wheat bran supplemented with either high nitrogen (soybean meal) or

low nitrogen (corn starch) C amendments to achieve four levels of amendment C:N ratios (10:1,

20:1, 30:1, and 40:1; Table 1). A non-amended, untreated control treatment was also included.

The total rate of added C in each amendment mixture was maintained at 4 mg C g-1

of soil. The

amendment nutrient analysis was done by Agricultural and Environmental services and

Laboratories, University of Georgia. The experimental design was a completely randomized

design with 4 replications, and the experiment was repeated. The relative biological availability

of C in amendments, soil, and sand was assessed by determining cold water extractable C and

hot water extractable C with modification of the procedure described by Ghani et al. (2003).

Briefly, 2 g of C amendment samples or 4 g of soil or sand samples were extracted in 40 mL of

deionized water for 30 min on rotational shaker at 20°C for cold-water extraction. For hot water

extraction, 40 mL of deionized water was added to sediments obtained after cold-water

extraction and incubated in a water bath at 80°C for 16 h before centrifugation to obtain extract.

Total organic C in water extracts was measured by acidification and sparging method to

eliminate inorganic carbon using a total organic carbon analyzer (TOC-VCPH model, Shimadzu,

Kyoto, Japan).

61

2.2 Experimental procedure

An equal amount of sand and soil containing C amendments was mixed by hand and used to fill

2,600-cm3 pots (12-cm diameter, 23-cm height). Six yellow nutsedge tubers were buried in each

pot, three at 5-cm, and three at 15-cm. Pots were saturated with tap water to fill soil pore space.

Oxidation-reduction potential (ORP) electrodes and temperature-moisture sensors (Combination

ORP Electrode, Sensorex Corp., Garden Grove, CA, USA and 5TM Soil Moisture Probe,

Decagon Devices, Pullman, WA, USA) were inserted at 10 to 15-cm depth and immediately

covered with black polyethylene mulch (0.03175 mm), which was secured with heavy-duty

rubber bands, and incubated in the growth chamber for three weeks. The first trial treatment

period was 15 March to 6 April 2013 and the second trial treatment period was 8 to 29 April

2013.

2.3 Cumulative redox potential

Soil temperature, redox potential, and soil moisture were continually monitored and recorded

hourly during treatment using ORP electrodes and an automatic data logging system (CR1000

with AM 16/32 multiplexers, Campbell Scientific, Logan, UT) over the three-week ASD

treatment. Due to a limited number of temperature probes available at the time of the study, soil

temperature was monitored only in 20 randomly selected pots while ensuring each treatment was

monitored in two replicate pots. Cumulative soil anaerobic activity was calculated as described

in Butler et al. (2012b). The data logging system provided raw soil redox potential (RP) values

on hourly basis and critical redox potential (CRP) was calculated as (CRP = 595mV –

(60mV*soil pH ). The absolute value of the difference between CRP and RP value was

determined for each RP. Cumulative soil anaerobic activity was then calculated by summing

absolute values over the three-week ASD treatment period.

2.4 Soil properties

At the end of ASD treatment, probes were removed, and a soil sample (~80 g wet wt.) was

collected using a clean plastic spoon from 0 to 8-cm depth of each pot. Subsamples were oven-

dried (105°C for 48 h) to determine gravimetric moisture content and the remaining sample was

62

air-dried and sieved (<2 mm). Soil pH was determined on air-dried samples in 0.01 M CaCl2

(1:2) using a pH electrode (Orion 3-Star Plus pH Benchtop Meter, Thermo Scientific, Waltham,

MA, USA) and are reported as soil pH determined in deionized water by adding 0.6. For

inorganic soil N and total soil N and C, 5-g of air-dried, sieved (<2 mm) soil was extracted with

1-M KCl for 30 min, centrifuged, and filtered (Whatman 42) prior to colorimetric analyses for

NH4-N and NO3-N + NO2-N using a microplate spectrophotometer (Powerwave XS, Biotek,

Winooski, VT, USA) as described by (Sims et al., 1995). Air-dried, sieved (<2 mm) and

pulverized soil samples were analyzed for total N and total C by flash combustion (Flash EA

1112 NC Soil Analyzer, Thermo Scientific). For extractable soil P, 5-g of air-dried, sieved (<2

mm) soil was extracted with Mehlich I extractant for 5 min (Mehlich, 1953), centrifuged, filtered

(Whatman 42) and extractant was determined using a malachite green microplate method

(D'Angelo et al., 2001).

2.5 Yellow nutsedge survival assessment

After ASD treatment, pots were incubated in the growth chamber and hand irrigated with tap

water supplied regularly throughout the growing period. Each pot was fertilized with 0.2 g N (as

blood meal) after ASD termination. After an 8-week period following soil treatment, yellow

nutsedge tuber mortality and biomass were assessed. Nutsedge roots with tubers were washed,

numbers of newly formed tubers were categorized as small (<0.5-cm ) and large (>0.5-cm) based

on average dry diameter and counted. Any non-germinating nutsedge tubers were recovered

from the soil and assayed for germination potential. The visual inspection of viable tubers was

completed by dissecting the tubers and then examining the internal color and condition of tubers

(Stoller and Wax, 1973). Ratings were assigned based on a 1 to 5 scale, 1 being firm and

undecomposed and 5 being completely rotten/decomposed with outer covering only. The cream

colored tissue of tubers with solid texture are viable tubers (Banks, 1983). Both root (including

tubers) and shoot biomass of plants were recorded after oven drying at 65°C for 48 h.

2.6 Organic acid assay

Organic acid production using carbon amendments with four C:N ratio during 7 day and 14 day

time course were carried out in the growth chamber separately in 9-cm tall pots. A potting

63

mixture was prepared similarly as described above. Three previously soaked yellow nutsedge

tubers were buried at 5-cm depth with IRIS tubes in each pot, which were irrigated until water

from pot’s bottom is discharged and then covered with polythene. Experiment design was

completely randomized design with three replications. On the 7th

day and the 14th

day, soil from

pots were mixed well and 20-30 cc of moist soil was placed in 50 ml centrifuge tubes to which

20 ml of 1 M KCl was added. Tubes were shaken for 30 min, at 180 rpm and centrifuged for 30

min, at 3500 rpm at room temperature. Supernatant (~ 10ml) was collected using 0.45 um

membrane filter, stored, and refrigerated in 20ml scintillation vials until analysis.

Accumulated organic acids were analyzed using a high-performance gas chromatograph

(Shimadzu GC-2010, Shimadzu, Kyoto) equipped with (Carbopack, B-DA/4% Carbowas 20M

columns). The liquid carrier consisted of helium, 24 ml/min. The 0.25 ml of internal standard 50

ppms of trimethylacetic acid in 0.03 M oxalis acid with ~ 25% meta phosphoric acid was mixed

with 1 ml of sample extract and 1 ul of supernatant was injected into column for analysis. We

analyzed acetic, butyric, isobutyric, methyl butyric, propionic, valeric, and isovaleric acids.

2.7 Statistical analysis

Data were analyzed with Mixed Model Analysis of Variance (MMAOV) macro (Saxton and

Auge, 2014), and Fisher’s P-LSD at p=0.05 using SAS version 9.3 (SAS Institute Inc., Cary,

NC). Repeated trials were treated as a random factor, whereas C amendments and C:N ratios

were treated as fixed factors for randomized completely block design factorial analysis. Data

were analyzed separately by C amendment to compare treatments with untreated control and also

by C:N ratio to compare with untreated control. Data were checked for normality and

homogeneity of variances. The data were rank transformed for non-normal data and unequal

variances separately and untransformed means and standard error of mean are reported.

3. Results and Discussion

3.1 Soil temperature and moisture

Soil temperature at 15-cm depth showed overall average temperature of 25°C. The mean soil

temperature generally ranged from a low of 15 to a high of 30°C during the treatment period and

64

was consistent in both trials. Soil moisture content was similar among carbon amended pots and

non-amended pots in both trials. Average gravimetric soil moisture at the beginning of trials was

0.08 g g-1

and at the end of ASD treatment soil moisture content increased to 0.23 g g-1

. Relative

increase in moisture in all treatments (65%) and fluctuating temperature in our experiment

should have significant effect on tuber germination and growth. Yellow nutsedge is reportedly

found in abundance in flooded soil (Ransom et al., 2009) and presence of moisture helps to break

the dormancy of buds. Usually, diurnal temperature fluctuation below 10°C creates problems

under plasticulture systems as nutsedge tubers actively sprout around 15 to 23°C (Stoller and

Woolley, 1983; Stoller and Sweet, 1987; Daugovish and Mochizuki, 2010) and nutsedge shoots

perforate plastic mulch, negatively influencing the efficacy of any disinfestation process under

plastic covering (Chase et al., 1999). During the three-week treatment period, some shoot

emergence through plastic was observed in all covered treatments (31 to 36% emergence). The

perforation is due to the sharp leaf tip emerging from germinated tubers placed in saturated soil

(Li et al., 2001) and some air trapped inside plastic. The 13-hour photoperiod maintained in the

growth chamber may have resulted in enhanced rapid rhizome differentiation (Stoller and

Woolley, 1983).

3.2 Cumulative anaerobic activity and soil pH

There was no interaction between C amendments and C:N ratios observed for cumulative

anaerobic activity and soil pH. The cumulative anaerobic activity significantly differed between

amended pots and the control pots with the lowest cumulative soil anaerobic activity observed in

the control (46,777 mV h; p=0.01). Among C amendments and C:N ratios, generation of

anaerobic conditions was statistically similar with average cumulative anaerobic activity of

110,175 mV h (Figure 2-1). Soil pH did not differ among C amendment treatments and the

control at treatment termination. However, soil pH differed (p<0.01) among C:N ratios with the

lowest numerical value in C:N ratio of 10:1 (pH 6.12; Figure 2-2).

Effectiveness of ASD for pathogen control is associated with anaerobic condition and lower pH.

As soil continues to deplete oxygen in saturated covered soil due to decomposition of

amendment by microbial activity, the anaerobicity is believed to be increased. In this regard high

anaerobicity generated in our treated pots which was similar to several ASD studies (Butler et

65

al., 2012a; Shrestha et al., 2013; McCarty et al., 2014), indicatimg that microbial response to

addition of amendments was higher. Treated pots could have stimulated the growth and

multiplication of anaerobic bacterial species (Mowlick et al., 2013b) rather than the fungal

population (Rosskopf et al., 2014b). Previous studies on ASD soil pH indicated that soil pH

decreased due to the release of organic acids from the anaerobic breakdown of added carbon. All

our treatments had lower soil pH than initial average soil pH (6.8) which may be due to the

production of acetic acid and butyric acid (Momma et al., 2006; Huang et al., 2015) and the

organic acid content may be higher for C:N 10:1, although organic acid extraction was not

performed. Surprisingly, lower pH and a slightly anaerobic condition in our non-amended

control suggest that some C required for microbial activity was already present in the soil. This

was revealed from our water extracted C in the soil:sand mixture which showed cold and hot

water extracted C rate of 2.76 mg kg-1

and 31.49 mg kg-1

respectively.

3.3 Soil nutrients and C:N ratio

Total soil inorganic N (i.e., NH4-N, NO3-N, and NO2-N) was significantly affected by treatment

(p = 0.001). Throughout this study, following ASD treatment, the lowest mean total soil

inorganic N was observed from the C:N ratio of 40:1 (lower than 4 mg N kg-1

soil). Total soil

inorganic N was primarily comprised of NO3 + NO2-N (76 to 91% of total inorganic N; Table 2)

rather than NH4-N (9 to 24%). The non-amended control had the least amount of soil NH4-N.

There was no significant interaction between C:N ratios and amendment type for soil nitrogen

content. However, there were significant differences in total soil inorganic N among C:N ratio

treatments (p<0.01; Table 2-2). Soil C:N ratio prior to treatment was (11.7). After termination of

ASD, the highest soil C:N ratio was observed for control (12.2) and least for treatment with C:N

10:1 (10.3). It was not surprising that total soil inorganic N was highest for 10:1 (60 mg) and

lowest for 40:1 (3.4 mg) given that soil C:N ratio was pre-adjusted. More mineralization of

amendments may have occurred at lower C:N ratio releasing high soil NH4-N but the overall soil

NH4-N was lower than that reported by Butler et al., (2012a). Nevertheless, our NO3 + NO2-N

was fairly higher and total N was comparable to that in a study conducted by McCarty (2014) for

C:N 15:1. We assume that soil N being measured on air-dried soil, the nitrification process

increased NO3 + NO2-N after ASD treatment assuring beneficial impact of ASD on nitrifying

66

bacteria (McCarty et al., 2014). Further, there may be microbial immobilization of available soil

inorganic N at higher C:N ratio, thus lowering total N.

Unlike soil N, Mehlich1 extractable soil P was significantly higher in wheat bran treatments than

molasses treatments and the non-amended control (p<0.0001). Among the C:N ratios, the

Mehlich-1 extractable soil P ranged from 17 to 28 mg P kg-1

soil and the highest values were

observed for C:N ratios 10:1 and 20:1. There was a significant interaction of C amendments with

C:N ratio for soil P. In treatments with wheat bran, the soil P was significantly higher at C:N

ratios 10:1 and 20:1, while soil P did not differ among C:N ratios maintained for molasses which

was similar to the study conducted by (Butler et al., 2012a; Butler et al., 2014a). The higher

value of Mehlich1 extractable soil P for wheat bran at lower ratio is due to high P content (81 to

112 mg/kg) of amendments. The dry molasses amendment mixture had significantly higher

levels of other nutrients besides P (8 to 32 mg kg-1

of amendments; Table 2-2) compared to the

wheat bran mixture.

3.4 Tuber germination

Several studies have confirmed that nutsedge tuber germination is not correlated to anaerobicity;

however, after ASD implementation, buried tubers were controlled by the method (Muramoto et

al., 2008; Butler et al., 2012a; McCarty, 2012). Moreover, buried tubers at greater depth (15-cm)

showed limited germination when compared to tubers deeper in the soil (Muramoto et al., 2008)

but, here two different burial depths (5 and15-cm) did not result in differences in germination. At

the shallower depth of 5-cm, tuber germination did not differ significantly among C treatments

but was higher in the non-amended control (77%; Figure 2-3). The germination of tubers at the

15-cm depth differed significantly among C treatments with lower percentage of germination for

wheat bran (29%) and C:N ratio 10:1 resulting in the lowest percentage of germination (33.3%)

and highest for the untreated control (81.3%). It was not surprising that our germination

percentage for dry molasses (65%) at greater depth was not comparable to the results of

(Muramoto et al., 2008; Butler et al., 2012a) as soil temperatures in those studies were higher

than those used here.

67

Nevertheless, the percentage of non-germinated tubers was significantly higher for wheat bran

(p<0.05; 86%) treatments compared to dry molasses and the control. Visual inspection of non-

germinated tubers based on their internal state showed that tubers turned to grey to black (rating

= 5) when they are totally decomposed and red, yellow, to brown when they were soft and in

various states of decomposition (rating = 4-3); and white (rating = 1) when firm and

undecomposed. The mortality of tubers in the wheat bran treatments is attributed to a higher

number of rotted tubers (scale of 4) and slightly decomposed tubers (scale of 2) which were

significantly greater than those harvested from dry molasses and control treatments (p<0.05;

Table 4).

3.5 Nutsedge growth and reproduction

Yellow nutsedge growth was significantly affected by C amendment and C:N ratio without any

interaction. Dry biomass of shoots and roots was greatly reduced by wheat bran compared to the

non-amended control. Between C amendments, the greater shoot and root biomass was recorded

for molasses. Among C:N ratios, both shoot and root biomass was lower for C:N ratio of 20:1;

however, the difference was not statistically significant for shoot biomass (Figure 2-5, 2-6). The

higher root biomass in the non-amended control and C:N 40:1 is in line with the production of

new tubers. Though no interaction was observed between C amendment type and C:N ratio on

mean tuber production, the number of tubers per pot was significantly higher from the control

treatment (33) and the lowest for wheat bran (13). Among total tubers, 79% of tubers were large

(>0.5-cm) in size and 21% small (<0.5cm) in size. The number of small tubers was highest for

40:1 and large tubers were highest in the non-amended control. Among C:N ratios, the number

of both large and small-sized tubers was lowest for C:N ratios 10:1 and greatest for 40:1 (Figure

2-4). The total tuber production was synchronous with the dry root biomass except C:N 10:1. It

was not surprising that both dry root biomass and tuber production were lower at C:N 10:1 ratio

as higher soil N content at this ratio promotes vegetative growth by enhancing basal bulb

formation rather that tuber formation (Garg et al., 1967; Stoller and Sweet, 1987). Also organic

acid, volatile fatty acids (VFA) and other toxic products may have led to biotransformation in the

soil, directly influencing tuber production and germination (Huang et al., 2015). Though we

maintained N fertility of soil after ASD treatment, only dry molasses enhanced the shoot growth

as compared to wheat bran confirming shoot growth is highly dependent on nutrient content of

68

applied amendments (see Table 2-2) rather than supplemental application of N fertilizer (Ransom

et al., 2009; McCarty et al., 2014). Production of acetic acid during amendment decomposition

did not seem to impact nutsedge sprouting (Ozores-Hampton et al., 1999). As N availability

becomes limited, allocation of nutrients is made to tuber production (Chellemi et al., 2013)

which may account for the larger number of tubers seen produced in the higher C:N (>10:1)

ratios.

3.6 Organic acid assay

Although ASD is a proven technique for pathogen control, in the case of weeds, particularly with

yellow nutsedge, complete eradication may be unattainable. Tubers buried in plastic-covered

pots produced 17% more tubers per m2 while amended pots showed only 0.4% increase in tuber

production. Although, temperature may be a limiting factor with regard to complete tuber

mortality, ASD treatment did significantly reduce tuber density, which can prevent crop yield

loss over time. The presence of high total soil inorganic N from amendment incorporation at the

lower C:N ratio (<30:1) could increase relative availability for crop uptake, thus reducing

fertilizer application, but nutsedge interference with the crop due to high denitrification (Volz,

1977) after ASD treatments could possibly interfere with crop yield. However, field application

of the target ratios would enhance understanding of this potential interaction.

At different C:N ratios, acetic, butyric and isobutyric were the primary organic acids observed in

soil solution during treatment which coincided with past studies (Momma et al., 2006; Rosskopf

et al., 2015). Total organic acids in soil at 7 and 14 days post treatment initiation were highest at

an amendment C:N ratio of 40:1 (Figure 2-7). The acid production was significantly higher using

dry molasses or wheat bran as ASD amendment during 7 days post treatment than in 14 days

post treatment (Figure 2-8). We did not observe any tuber germination or decomposition in

amended pots. We believed that our experimental period i.e. 7 days and 14 days was not enough

to cause any significant impact on tuber decomposition.

69

4. Conclusion

Overall, the use of wheat bran as an ASD amendment, applied at lower C:N ratio, provided

better tuber control than dry molasses. These amendments not only differ in their nutrient

content, but also differ in decomposition rate i.e. dry molasses has more sucrose content making

it sticky, leachable during treatment application, and results in faster decomposition (Stock,

2008) while wheat bran is more fibrous, absorbing more water and decomposes more slowly

(Stevenson et al., 2012). As described earlier, in ASD, microbial decomposition of these

amendments, associated with increases in Clostridia and Bacilli spp. (Mowlick 2012; 2013)

plays an important role in the production of VFA and organic acids. The concentration of acids

produced during ASD may not be high enough for decomposition of tubers alone, as the

concentration of acetic acid needed to effectively kill 1- to 4-week old sprouted nutsedge tubers

is 30% (Abouziena et al., 2009). It is more likely that multiple mechanisms are at work. Adebajo

(1993) reported that yellow nutsedge tubers contain sucrose and have inhabiting microbes such

as yeasts, Pseudomonas, and Bacillus spp. In the same study, tuber tissue was found to inhibit

some microbial growth. Microbial community analysis of tubers is currently underway to

ascertain the potential roll of biological degradation of tuber tissue. It is possible that extended

ASD treatment might increase weed suppression and this will be investigated in the future.

70

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78

Appendix

Table 2-1. Amount of C amendments for different C:N ratios at 4 mg C g-1

of soil for each pot

Treatments

C amendment Rate of amendments

ratio percentage g kg

-1 of soil

C:N C N CN10 CN20 CN30 CN40

Dry molasses

DM 29.7 38.7 1.3 6.4 9.4 10.3 7.7

Soybean

meal 4.8 42.6 8.8 3.6 0.9 - -

Corn starch - 40.3 0.0 - - 0.1 2.6

Wheat bran

WB 13.3 41.7 3.13 7.8 6.4 4.2 3.2

Soybean

meal 4.8 42.6 8.84 1.8 - - -

Corn starch - 40.3 0.02 - 3.3 5.6 6.7

N.B. C amendments are mixed uniformly with 3 kg of soil: sand for each pot. CN10=C:N ratio

10:1, CN20=C:N ratio 20:1, CN30=C:N ratio 30:1 and CN40=C:N ratio 40:1, Dry molasses

(DM), Wheat bran (WB)

79

Table 2-2. Soil inorganic phosphorus, soil nitrite+nitrate-N (NO2+NO3-N) and ammonium-

nitrogen (NH4-N) and total inorganic N as affected by soil amendments and amendment C:N

ratios

Treatment

Soil nutrients *

Soil C:N ratio Inorganic P NO2 + NO3-N NH4-N

Total

inorganic N

Amendment mg nutrient kg-1

soil

Control 14 ± 0.8 b 4.7 ± 0 c 1.4 ± 0.2 b 6 ± 1.6 b 12.2 ± 0.1 a

DM 16.2 ± 0.9 b 19.6 ± 0 b 2 ± 0.2 a 21.5 ± 1.2 a 11.1 ± 0.2 b

WB 28.1 ± 2.2 a 22.7 ± 0 a 2.2 ± 0.2 a 25 ± 1.2 a 10.7 ± 0.2 b

p<0.001 p=0.0309 p=0.001 p<0.001 p<0.001

C:N ratio

CN10 28.3 ± 3.4 a 57.6 ± 0 a 2.3 ± 0.2 a 59.8 ± 1.7 a 10.3 ± 0.4 d

CN20 24.9 ± 3 a 19.3 ± 0 b 1.9 ± 0.2 a 21.2 ± 1.7 b 11.3 ± 0.1 c

CN30 18.4 ± 1.9 b 6.4 ± 0 c 2.1 ± 0.2 a 8.5 ± 1.7 c 11.6 ± 0.1 bc

CN40 17.1 ± 1.5 bc 1.4 ± 0 d 2 ± 0.2 a 3.4 ± 1.7 e 11.9 ± 0.3 ab

Control 14 ± 0.8 c 4.7 ± 0 c 1.4 ± 0.2 b 6 ± 1.7 d 12.2 ± 0.1 a

p<0.001 p<0.001 p=0.009 p<0.001 p<0.001

* Within columns values (mean ± SE) followed by different letters are significantly according to

Fisher’s Protected LSD test at p<0.05.

80

Table 2-3. Tuber ratings of non-germinated tubers for amendment and C:N ratio treatments

No. of

tubers

buried in

the

experiment

No. of non-

germinated

tubers*

No. of tubers for each rating scale

Treatments 5 4 3 2 1

Control 96 20 11 4 5 0 0

Dry molasses

10:1 48 25 8 7 7 2 1

20:1 48 18 9 7 1 0

30:1 48 7 3 4 0 0 0

40:1 48 13 7 3 3 0 0

Total 192 63 27 21 11 2 2

Wheat bran

10:1 48 28 10 5 5 2 6

20:1 48 26 8 7 7 3 1

30:1 48 31 8 14 6 2 1

40:1 48 28 14 6 4 3 1

Total 192 113 40 32 22 10 9

Total tubers 480 196

*Rating scale: 5- Completely rotten/decomposed with outer covering only, 4- Soft and

decomposed, 3- Slightly soft and decomposed, 2- Firm and slightly decomposed and 1- Firm and

undecomposed.

81

Bars indicated by different letters are significantly different, p<0.05. Error bars indicate standard

error with four replicates. Control=non-amended control, 10:1=C:N ratio 10:1, 20:1=C:N ratio

20:1, 30:1=C:N ratio 30:1 and 40:1=C:N ratio 40:1

Figure 2-1. Effect of amendment C:N ratio on mean cumulative anaerobic condition during

ASD.

a a a

a

b

0

20000

40000

60000

80000

100000

120000

140000

10:1 20:1 30:1 40:1 Control

Cu

mu

lati

ve

an

ae

rob

ic c

on

idti

on

(

mV

h)

82



20:1, 30:1=C:N ratio 30:1 and 40:1=C:N ratio 40:1

Figure 2-2. Effect of amendment C:N ratio on mean soil pH in CaCl2 at treatment termination.

c

a ab

ab b

5.3

5.35

5.4

5.45

5.5

5.55

5.6

5.65

5.7

5.75

5.8

5.85

10:1 20:1 30:1 40:1 Control

So

il p

H

83

Figure 2-3. Effect of amendment and amendment C:N ratio on mean percentage of tuber

germination per pot during ASD.



20:1, 30:1=C:N ratio 30:1 and 40:1=C:N ratio 40:1,m=mean, DM=dry molasses, WB=wheat

bran.

c

bc b bc

a

0

10

20

30

40

50

60

70

80

90

100

10:1 20:1 30:1 40:1 Control

Ge

rmin

ati

on

of

tub

ers

(P

erc

en

tag

e)

5 cm 15 cm mDM-15 cm mWB -15 cm mCtrl

84

Bars indicated by different letters are significantly different, p<0.05. Capital letters are used to

compare the respective means of large tuber production and small letters are used to compare

respective means small tuber production. Error bars indicate standard error with four replicates.

Control=non-amended control, 10:1=C:N ratio 10:1, 20:1=C:N ratio 20:1, 30:1=C:N ratio 30:1

and 40:1=C:N ratio 40:1,m=mean, DM=dry molasses, WB=wheat bran.

Figure 2-4. Effect of amendment and amendment C:N ratio on mean percentage of number of

tuber production per pot during ASD.

C

BC

BC B

A

c bc bc

a ab

0

5

10

15

20

25

30

35

10:1 20:1 30:1 40:1 Control

Nu

mb

er

of

tub

er

pro

du

cti

on

pe

r p

ot

Large tuber Small tuber mDM Large tuber

mWB Large tuber mCTRL

85



20:1, 30:1=C:N ratio 30:1 and 40:1=C:N ratio 40:1, m=mean, DM=dry molasses, WB=wheat

bran.

a

b b b b

0

2

4

6

8

10

12

14

10:1 20:1 30:1 40:1 Control

Dry

sh

oo

t b

iom

as

s (

g)

Shoot

Shoot mDM

Shoot mWB

Figure 2-5. Effect of amendment and amendment C:N ratio on mean percentage of dry shoot

biomass production per pot during ASD.

86



20:1, 30:1=C:N ratio 30:1 and 40:1=C:N ratio 40:1, m=mean, DM=dry molasses, WB=wheat

bran.

bc c

bc

ab

a

0

2

4

6

8

10

12

14

10:1 20:1 30:1 40:1 Control

Dry

ro

ot

bio

ma

ss

(g

)

Root

Root mDM

Root mWB

Figure 2-6. Effect of amendment and amendment C:N ratio on mean percentage of dry root

biomass production per pot during ASD.

87

Figure 2-7. Total organic acids present in soil at 7 and 14 days post treatment initiation.

Bars indicated by the same letters are not significantly different (p>0.05). Capital letters are used

to compare the respective means of total acid production at 7 days and small letters are used to

compare respective means of total acid production at 14 days. 10:1=C:N ratio 10:1, 20:1=C:N

ratio 20:1, 30:1=C:N ratio 30:1, and 40:1=C:N ratio 40:1, all at C rates of 4 mg C g-1

soil; U-

UTC=uncovered and untreated (non-amended ) control, CUTC=plastic covered and untreated

(non-amended ) control. Error bars represent standard error.

AB

AB

B

A

C C

b

cd

bc

a

d

d

0

2

4

6

8

10

12

14

16

18

20

22

24

26

28

10:1 20:1 30:1 40:1 U-UTC C-UTC

To

tal

Ac

id (

mm

ole

/kg

so

f s

oil)

7 days 14 days

88

Figure 2-8. Various organic acids present in soil at 7 and 14 days post treatment initiation.

10:1=C:N ratio 10:1, 20:1=C:N ratio 20:1, 30:1=C:N ratio 30:1, and 40:1=C:N ratio 40:1, all at

C rates of 4 mg C g-1

soil.

89

Chapter 3

Organic amendment type and C:N ratio impact on Fusarium

oxysporum f. sp. lycopersici following anaerobic soil disinfestation

90

A version of this chapter is a manuscript in preparation for Phytopathology by Utsala Shrestha,

Alex Bruce, Bonnie H. Ownley and David M. Butler.


analysis, results interpretation and writing. Alex Bruce helped in data collection of Fusarium

population from soil.

91

Abstract

Anaerobic soil disinfestation (ASD) relies on the incorporation of organic amendments to

provide labile carbon (C) to stimulate microbial activity in saturated soil mulched with

polyethylene. Two organic amendments, dry molasses and wheat bran, were incorporated

independently in soil:sand mixture in pots with ASD treatment to evaluate effectiveness of these

organic amendments at 4 mg C g-1

soil, with varying C:N ratios of 40:1, 30:1, 20:1 and 10:1

against introduced Fusarium oxysporum (Fo) propagules. Soil pH and cumulative anaerobic

condition were assessed to determine soil anaerobic condition. Similarly, a field study with dry

molasses as the primary C-source amendment, with the same four C:N ratios was carried out. In

addition, a C:N ratio of 30:1 at a lower C rate of 2 mg C g-1

soil, an untreated control, and a

MeBr-fumigated control were included. After three weeks of ASD treatment, Fo survivability

was assessed by dilution plating of recovered inoculum bags from the soil on Snyder-Nash agar.

Across both OAs, soil pH was least for the C:N ratio of 10:1, but there were no soil pH

differences among other treatments in the pot study. We did not observed any soil pH difference

before and after ASD treatment. For both OAs in the pot study and dry molasses in the field

study, cumulative anaerobic condition was greater (more anaerobic) than control treatment. Fo

colonies were fewer for dry molasses maintained at C:N ratio 20 to 30 than wheat bran in the pot

study. All dry molasses treated plots in the field study significantly suppressed Fo than in non-

amended plots. Our results suggest that application of C rates at 4 mg C g-1

soil for ASD

treatment induces more anaerobic soil conditions and greater mortality of Fo inoculum compared

to lower C rate (2 mg C g-1

soil), at a C:N ratio of 20:1 and 30:1.

Keywords: Anaerobic soil disinfestation, Fusarium oxysporum, organic amendments, pepper

yield

92

1. Introduction

Anaerobic soil disinfestation (ASD) is an anaerobically mediated pre-plant soil treatment

developed to control soilborne pathogens in high-value specialty crop production. ASD is one of

the substitutes to chemical fumigation that relies on the incorporation of organic amendments to

provide labile carbon (C) to stimulate microbial activity in saturated soil mulched with

polyethylene (Butler et al., 2012b). Suppression of various soilborne pathogens utilizing different

carbon amendments during ASD have been reported (Shennan et al., 2014) and compared

(Strauss and Kluepfel, 2015) showing its viability to replace the chemical fumigants. However,

results on suppression of different pathogens across diverse cropping systems are not common.

Even similar types of pathogens showed variation in suppression level during ASD treatment as

effectiveness of ASD is dependent upon chemical, physical and biological soil properties

(Rosskopf et al., 2015). To date, disease suppression due to ASD treatment is inferred as a

consequence of microbial shifts, production of organic acids (Momma et al., 2006), and other

volatile compounds like alcohols, organic sulfides, esters, ketones, hydrocarbons, and

isothiocyanates (Hewavitharana et al., 2014).

Fusarium oxysporum f. sp. lycopersici (Fol) is a persistent soilborne pathogen that causes wilt

and dry rot disease by producing pectin degrading enzymes (Jones et al., 1972) in tomato

(Solanum lycopersicum L.), resulting in great production losses (Walker, 1971; McGovern,

2015). After phase out of the ozone depleting fumigant methyl bromide, sustainable disease

management practice to control Fol is warranted along with environmentally friendly cultural

practices that enhance soil quality and microbial activity (Ristaino and Thomas 1997). ASD has

evolved as an alternative practice for methyl bromide and other chemical and non-chemical

fumigants where they are restricted due to various limitations such as registration issue or

economic and ecological limitations. ASD can be easily adapted to plastic culture system and

utilizes a broad range of organic amendments to generate anaerobic conditions in the tarped soil.

Incorporated organic amendments increase the organic matter in the soil making soil rich in

nutrients and pathogen suppressive (Bonanomi et al., 2013). The wide range of amendments used

in ASD have proven to control various host specific Fusarium oxysporum (Fo). More than 50%

mortality of Fo against non-amended covered control was achieved with various agriculture by-

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products (plant residue, cereal bran, mustard meal) cruciferous plant, grasses, cover crops (Blok

et al., 2000; Yossen et al., 2008; Butler et al., 2012a; Butler et al., 2012b; Mowlick et al., 2012a;

Mowlick et al., 2012b; Momma et al., 2013; Mowlick et al., 2013b; McCarty et al., 2014), and

liquid amendments like molasses, ethanol (Momma et al., 2011; Mazzola and Hewavitharana,

2014), bioethanol (Horita and Kitamoto, 2015), and organic acids (Shennan et al., 2014; Horita

and Kitamoto, 2015). The most common incubation period was 2 to 3 weeks with average soil

temperature greater than 20°C while maximum incubation period of 13 weeks was reported in a

study conducted by Blok et al. (2000) using rye grass, clipped grass and broccoli and a maximum

temperature of greater than 35°C reported in Florida using liquid molasses and broiler litter

(Butler et al., 2012a). Effective control of Fo f. sp. asparagi was observed in a study conducted

by Blok et al. (2000) and similar effects were obtained in Mowlick et al. (2013a) using mustard,

oat grass, radish and wheat bran to control Fo f. sp. spinaciae. ASD was also found to be

effective for banana wilt caused by Fo f. sp. cubense when 1% corn stalk was used in pot study

(Huang et al., 2015). So far, Fol is the most studied Fusarium pathogen with 50-99% reduction

in propagules under ASD pot and field treatment conditions. However, in the field situation

effectiveness is observed only when organic amendments applied were wheat bran or ethanol/

bioethanol or liquid molasses in combination with broiler litter or organic acid accompanied by

high temperature (>28°C). Under controlled pot or box environments, under moderate

temperature (>24°C), warm-season cover crops pearl millet, sunn hemp, sorghun-sudangrass,

cowpea alone or mixed with pearl millet or sorghum-sudangrass (Butler et al., 2012b), ethanol,

bioethanol, wheat bran (Momma et al., 2010; Horita and Kitamoto, 2015) effectively controlled

the Fol pathogen.

Previous results showed that wheat bran and molasses have significant positive effects in the

eradication of Fol. Wheat bran at the rate of 2 to 3 kg m-2

in (Mowlick et al., 2013a; Mowlick et

al., 2014) or at lower rate of 1 kg m-2

(Yossen et al., 2008; Momma et al., 2010; Mowlick et al.,

2012a) was found to reduce Fo inoculum in various soil types (sandy, loam and volcanic and

gray lowland soil). Mortality of both fungal spores and chlamydospores of Fol under wheat bran

is reported to occur with the production of acetic acid and butyric acid (Momma et al. 2005;

Momma et al. 2006). Only liquid molasses is applied as a carbon supplement to treat soil for Fol

disease (Momma et al., 2010; Butler et al., 2012a) and dry molasses has not been studied against

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Fol. Considering the efficacy of wheat bran and the availability of dry molasses, we selected

these as organic amendments to test effectiveness in controlling introduced Fol inoculum

following ASD treatment. Realizing the importance of carbon to nitrogen ratio (C:N) of these

amendments, we also sought to evaluate the effectiveness amendments at different C:N ratios.

Amendment C:N is widely reported to influence soil microbial activity, post-treatment nutrient

availability for crops following ASD treatments that ultimately affects yield of crops (Butler et

al., 2014a). The overall aim of this study was to i) evaluate the cumulative anaerobic soil

condition, followed by amendment application for ASD treatment and ii) evaluate the

effectiveness of wheat bran and dry molasses maintained at C:N ratios of 10, 20, 30 and 40 as a

carbon source amendment to suppress introduced inocula of Fol after ASD treatment.


2.1 Fusarium inoculum preparation

Fusarium oxysporum f. sp. lyscopersici as a target pest was isolated from diseased tomato plants

at the Organic Crops Unit, Knoxville, Tennessee, USA. The diseased plant parts were cleaned

with tap water and then surface disinfested using 10% commercial bleach solution for 1 min. The

plant was cut in 2.5-cm sections, plated on water agar medium (1.5%), and then incubated at 23

± 2°C until fungal growth was apparent from each plant part. After 3 days, the fungal mycelium

were isolated in antibiotic amended potato dextrose agar (PDA), amended with 10 mg liter-1

rifampicin (Sigma-Aldrich, St. Louis, MO) and 3.45 mg liter-1

fenpropathrin (Danitol 2.4EC,

Valent Chemical, Walnut Creek, CA), and re-isolated after several days to obtain pure isolates.

For inoculum production, 100 g of rice (Uncle Ben's® Whole Grain Brown Rice) was hydrated

in double deionized water for 24 h in a 500 ml Erlenmeyer flask. Excess water from flasks was

drained, autoclaved at 121°C for 55 min and shaken afterward to avoid any clumps. Ten 5- to 7-

mm plugs of mycelium from the actively growing edge of fungal colonies were placed inside the

autoclaved flask to inoculate the grains. Flasks were incubated at room temperature under

fluorescent light for 2 weeks to allow fungal growth and colonization of rice grains. The

colonized rice grains were air dried for 2 to 3 days under the fume hood on an absorbent under

pad and used immediately or stored in airtight zip lock bags at 4°C for preparation of pathogen

inoculum packets. About 2 g of dehydrated rice grain inoculum was sealed in 5-cm × 5-cm

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packets made of apertured Delnet® polyolefin fabric (DelStar Technologies, Austin, TX).

Strings were attached in each packet for easy removal for both pot and field experiments.

2.3 Pot experiment

The experimental design for the pot study was completely randomized, with a 2 × 4 factorial,

with organic amendments and C:N ratios as the main effects. As described above, dry molasses

(C:N~29.7, Westway, New Orleans, LA) and wheat bran (C:N~13.3, Siemer milling company)

were selected as carbon sources. For C:N ratios, dry molasses and wheat bran were adjusted to

four ratios of 40:1, 30:1, 20:1 and 10:1 at 4 mg of C g-1

soil using soybean meal (C:N~4.8, Hi

Pro Soybean meal, Fiona, TX) and/or corn starch (C:N~0, Tate & Lyle ingredients Americas,

INC. Decatur, IL). Each mixed amendment was well incorporated into sieved soil (<10 mm,

Dewey silt loam, total carbon = 0.94%, pH = 6.2, and collected from University of Tennessee,

Organic Crops Unit, Knoxville, TN) and fine sand (50:50). The control without organic

amendment was included for both dry molasses and wheat bran and the study was repeated. The

first trial started in June 12, 2013 and the second in July 8, 2013. Soil was collected from

different locations each time. Initially the soil sand mixture had a C:N ratio of 11.6 and 12.4, and

a mean cold water extractable C of 2 and 3.1 mg k g-1

, and a total N of 3.3 and 2 mg k g-1

of soil

in trial 1 and trial 2, respectively.

Polyethylene black pots (size 12-cm diameter and 23-cm height) were filled with the amendment

soil and sand mixture and two Fusarium inoculum packets, were buried at 5- and 15-cm depths

in each pot. Oxidation-reduction electrodes (ORP) and temperature-moisture sensors

(Combination ORP Electrode, Sensorex Corp., Garden Grove, CA, USA and 5TM Soil Moisture

Probe, Decagon Devices, Pullman, WA, USA) were inserted at 10- to 15-cm depth to measure

cumulative redox potential and temperature of soil. Each treatment with four replicates received

ORP probes, but only two replicates received temperature probes. Pots were filled with tap water

until water flowed from the bottom of pots to attain complete saturation. Pots were covered with

black polyethylene (0.03-mm), held in place with heavy-duty rubber band and arranged in the

growth chamber, which was maintained at 25°C for 14 h and 15°C for 10 h, with 50% relative

humidity for 3 weeks.

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At completion of ASD, ORP were removed, and a soil sample (~80 g wet wt.) was collected

using a clean plastic spoon up to 8-cm depth of each pot. Subsamples were oven-dried (105°C

for 48 h) to determine gravimetric moisture content and the remaining sample was air-dried and

sieved (<2 mm) to measure soil pH. Soil pH was measured in 0.01 M CaCl2 (1:2 soil to solution

ratio) using a pH electrode (Orion 3-Star Plus pH Benchtop Meter, Thermo Scientific, Waltham,

MA, USA) and values were adjusted to soil pH determined in deionized water by adding 0.6

units. Cumulative soil anaerobic condition was calculated as described in Butler et al., (2012b)

and McCarty et al., (2014).

2.3.1 Assessment of Tomato plant

Three-week-old seedlings of dwarf patio tomato ‘Florida Lanai’ variety were planted in each pot

after removal of Fol packets at the termination of ASD treatment to evaluate plant growth

characteristics. After approximately 8 weeks, plants were removed, scored for diseases and total

dry biomass determined.

2.4 Assessment of Fusarium population

After ASD termination, Fusarium populations were assessed by standard dilution plating of

recovered inoculum onto Nash-Snyder medium1(5 g Difco Peptone, 1 g KH2PO4, 0.5 g MgSO4

7H2O, 20 g Agar, 1 g Pentachloronitrobenzene) (Nash and Snyder, 1962). After two weeks,

identification of Fol colonies were confirmed by microscopic examination of morphological

features, and colony forming units were counted.

2.5 Field experiment

2.5.1 Layout treatment establishment

The field study was established at the UT Plateau Research and Education Center in Crossville,

TN on May 16, 2013 and May 13, 2014 to assess the ASD effect on Fusarium populations. The

design was a randomized complete block design with four replications. Each plot size was 7.6 m

× 1.8 m. The soil type is a Lily series (Fine-loamy, siliceous, semiactive, mesic Typic

Hapludult), which was tilled just to remove any debris. Soil amendments were applied in each

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plot using a drop fertilizer spreader and were thoroughly incorporated with a rotovator. Raised

beds (~5-cm) were formed, mulched with standard black polyethylene, and drip irrigated to fill

pore space. Anaerobic soil conditions were monitored using iron oxihydroxide-coated tubes,

which were installed after applying plastic mulch and before irrigation (Castenson and

Rabenhorst, 2006). Dry molasses at a total C rate of 4 mg C g-1

soil was used as the primary C

source amendment at four C:N ratios of 10:1 (ASD10), 20:1 (ASD20), 30:1 (ASD30) and

40:1(ASD40) and the application rate was 0.86, 1.26, 1.38 and 1.03 kg m-2

respectively. The C:N

ratios of 10:1 and 20:1 were adjusted using soybean meal (0.48 and 0.12 kg m-2

respectively) and

C:N ratios 30:1 and 40:1 were adjusted using corn starch (0.01 and 0.35 kg m-2

respectively). In

addition, a C:N of 30:1 at a lower C rate of 2 mg C g-1

soil (LCASD) with 0.69 kg m-2

dry

molasses and 0.01 kg m-2

of corn starch were included. A non-amended untreated control (UTC)

and a methyl bromide (MeBr) fumigated control (67:33 mixture with chloropicrin, 200 lbs acre-1

)

were also included. Research was conducted in different sites each year. In 2013, soil had initial

gravimetric soil moisture content of 2.5 to 2.7 g g-1

, soil C:N of 12.9, total N of 6.5 mg N kg-1

soil and 4.9 mg P kg-1

soil. Initial gravimetric soil moisture content in 2014 was 2.7 to 3.1 g g-1

with soil C:N of 13.38, total N of 6.8 mg N kg-1

soil and 9.3 mg P kg-1

soil.

2.5.2 Anaerobic soil conditions and Fusarium assay

To measure the anaerobic condition of soil two iron oxihydroxide coated IRIS tubes (polyvinyl-

chloride pipe) were inserted per plot (Castenson and Rabenhorst, 2006). Reduction of iron (Fe)

was assessed as described by Rabenhorst (2012). In 2014, along with IRIS tubes, the anaerobic

condition was assessed inserting two ORPs in each plot of one block and soil temperature was

recorded with datalogger temperature probes. Due to unavailability of datalogger temperature

probes in 2013, hand held temperature probes were inserted in the soil to measure soil

temperature. Fusarium packets were prepared as describe above for the pot study. Two packets

containing propagules of Fol were buried at a 10- to 15-cm depth in each bed in 2013 and two

additional inoculum packets were buried in 2014. Due to logistical constraints, Fo inoculum

packets were not introduced into the MeBr fumigated plots. At the end of the ASD treatment (3

weeks), inoculum packets were retrieved and Fol propagule survival was assessed similarly as in

the pot study. Soil samples (10 composite cores to 15-cm depth) from each plot were collected

before applying amendments (preASD), at ASD termination (endASD), and 3 weeks following

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ASD treatment termination (postASD) to access soil moisture content, and endemic Fusarium

populations. Soil samples for gravimetric moisture content was assessed at 105°C immediately

after collection and soil samples for Fusarium assay soil were kept at 4°C until analysis.

Fusarium populations were assessed similar to Fo packet inoculum after soil samples were dried

(24 h) and used for serial dilution.

2.5.3 Crop performance and root disease assessment

On June 10, 2013, bell pepper transplants (cv. Aristotle F1) were planted 30.5-cm between and

within a double row per bed (30 plants per bed) to assess crop performance, plant nutrition, and

incidence of disease. Weed populations and incidence of soilborne disease were monitored

throughout the growing season at least three times. Plant heights were measured each week in

July beginning three weeks after transplanting. At first harvest (after 9 weeks) 20 recently mature

leaves were taken from each treatment, dried at 65°C, ground and total N and C were determined

by combustion (data not shown). Three root systems were taken randomly at the end of harvest

to evaluate root galling and condition. Ratings of galling by root-knot nematode were based on

Bridge and Page (1980), with the extent of root galling present on bell pepper plants rated as

follows: 0 = no nematodes, 1 to 4 = galling of secondary roots only, 5 to 10 = galling of primary

laterals and tap root. Each root system was also evaluated on a scale of 1 to 5 (1 = brown with

signs of decay; 5 = white and healthy) that represented health status of the root system.

Peppers were harvested and graded according to standard USDA fruit grading; Fancy, No. 1, No.

2, and cull (USDA-AMS, 2005). Bell pepper were harvested once a week during mid-to-late

August. Peppers were harvested based on size and dark green color and firmness. Fruit were

harvested from the middle (~26 plants) except plants at the end of each row. Fruit were counted

and weighed in each grade class. For culls, reason for culling and number of fruit culled were

recorded.

2.6 Statistical analysis

Mixed model analysis of variance was conducted with SAS (9.3 SAS Institute, Cary, NC); data

were checked for normality and homogeneity of variances, and transformed as needed. Least

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squares means were compared with Fisher's P-LSD at 5% significance level and untransformed

means are reported. Relationships between cumulative anaerobic conditions and Fusarium

colony counts were assessed using correlation analysis at p<0.05. Data were analyzed separately

by C amendment to compare treatments with untreated control and also by C:N to compare with

untreated control. Fusarium inoculum survival data was collected as colony forming unit per

gram of soil and transformed using the formula log10(x+1) before statistical analysis.

3. Results

3.1 Pot study

No apparent relationship between cumulative anaerobic condition, pH and Fusarium population

was observed in trial 2 and only moderate negative correlation between cumulative anaerobic

condition and Fusarium population was observed in trial 1 (-0.4, p<0.02). The weak negative

relationship between soil C:N at the end of ASD treatment and Fusarium population was

observed in trial 2 (-0.34 , p=0.03). At the end of treatment, the soil sand mixture irrigated and

covered had 2.4 mg kg -1

of NH4N, 24.4 mg kg -1

of NO3NO2, 5.8 mg kg -1

of inorganic P,

and10.1 C:N in trial 1. Whereas in trial 2 the mixture had 5 mg kg -1

of NH4N, 24.4 mg kg -1

of

NO3 +NO2, 6.8 mg kg -1

of inorganic P, and 12.1 C:N.

3.1.1 Gravimetric soil moisture, soil temperature, and soil pH

Gravimetric soil moisture content before ASD treatment initiation was 0.11 g g -1

for trial 1 and

0.18 g g -1

for trial 2. After 3 weeks, average soil moisture content increased by 0.53% for trial 1

and 0.26% for trial 2. The gravimetric soil moisture content was higher for amended pots than

control pots in both trials. However, volumetric soil content did not differ across organic

amendment and C:N treatments (Table 1). Mean soil temperature across C:N ratios ranged from

23.7 to 24.7°C in trial 1 and 23.3 to 24.4°C in trial 2 and was significantly higher in organic

amendment treated soil (23.9 to 24.1°C) than control (23.3°C) only in trial 2. Soil pH did not

differ among organic amendments and control in the both trials, and there was no interaction of

organic amendment and C:N. However, across C:N ratios, soil pH was lowest for the 10:1

(Figure 3-1A, 2B).

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3.1.2 Anaerobic soil conditions

There was a significant effect of organic amendment, C:N on cumulative soil anaerobic

conditions (p<0.001) as well as a significant interaction of organic amendment with C:N

(p<0.03) on both trials. For organic amendments, mean cumulative soil anaerobic conditions

were higher (i.e., more anaerobic) for dry molasses (222,780 – 233,098 mV h) than wheat bran

(179,338 – 205,104 mV h) and lowest for the control (142,324 – 92,823 mV h). In treatments

with dry molasses, cumulative anaerobic conditions were lowest at C:N 40:1 in both trials; while

amendments with wheat bran showed lowest cumulative anaerobic conditions at 20:1 C:N and

30:1 C:N for trial 1 and trial 2, respectively. The greatest difference between organic

amendments in accumulation of anaerobic conditions was observed for 20:1 C:N (72,469 mV h;

Figure 3-2A) and 30:1 C:N (67,662 mV h; Figure 3-2B).

3.1.3 Fusarium population

Across all amended treatments, Fol populations were significantly lower at the 15-cm depth than

at 5-cm (19% in trial 1 and 35% in trial 2), but there were no interactions of depth with organic

amendment or C:N ratios in both trials. Fol counts were lowest in control treatment at 15-cm

depth in trial 2 (Table 2). There was a significant interaction of organic amendment and C:N in

Fol counts in trial 1 (p<0.001), with fewer Fol colonies recovered from the lower C:N ratios

(10:1, 20:1 and 30:1) for dry molasses compared to the C:N 40:1 and the control (2.6 to 2.7 log10

[CFU+1] g -1

). There was no difference among C:N ratios and the control for wheat bran and

mean Fol counts ranged from 3.5 to 3.7 log10[CFU+1] g -1

(Figure 3-3A). We did not observe

any interaction of organic amendment and C:N and differences in trial 2. Nevertheless fewer Fol

colonies were observed in control and dry molasses treated pots, especially in the 10:1 and 20:1

(2.8 and 2.6 log10[CFU+1] g -1

respectively) than wheat bran treated pots (3.7 log10[CFU+1] g -1

,

Figure 3-3B).

3.2 Field study

During the treatment period, mean soil temperature was 22.04 to 24.7°C in 2013 and 23.6 to

24.5°C in 2014. In 2013, at termination of ASD the gravimetric soil moisture content ranged

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from 1.5 to 2.9 g g -1

across treatments with highest moisture content observed for ASD20 (3.1 g

g -1

) and lowest for fumigated plot (1.5 g g -1

). In 2014 soil moisture content ranged from 2.9 to

3.9 g g -1

, and was highest for ASD30 (3.9 g g -1

). Soil pH, as in the pot study, did not differ

among organic amendments and control in both years. However, soil pH change was higher after

three weeks of ASD implementation in 2013 (0.51 to 0.64 units) than in 2014 (-0.01 to 0.06

units). Similar to trial 1, no significant relationship between oil pH, anaerobic condition and

Fusarium population was observed.

We did not observe any galling during field trials. The total number of pepper fruits was

significantly higher in amended plots with lower C:N (10 and 20) than fumigated plots and non-

amended plots. However, this difference was seen only in the year 2014. Marketable and fancy

fruit numbers per hectare was higher in all amended plots with 4 mg C g-1

soil (Figure 3-6).

Similarly, total fruit yield (61 to 71 mt ha-1

) and marketable yield (54 to 61 mt ha-1

) was

significantly higher in the second year in amended plots than in fumigated and non-amended

plots (<50 mt ha-1

).

3.2.1 Anaerobic soil conditions

Across the treatments, the percentage of oxihydroxide paint removal was higher for all amended

treatments with 4 mg C g-1

of soil in both years than non-amended control, indicating enhanced

anaerobic conditions driven by soil microbial activity. Percentage of oxihydroxide paint removal

in reduced C rate amended treatment (LCASD30) was intermediate between high C rate

amended plot and non-amended plot. However, cumulative anaerobic soil condition of

LCASD30 was found similar to high-amended treatments (p<0.002, Figure 3-4). The

accumulated anaerobic condition in 2014 also revealed a similar trend across all treatments with

the lowest mean anaerobic condition recorded for control plots (105,737 mV h).

3.2.2 Fusarium population

Inoculum packets retrieved from soil after ASD termination showed no differences in population

count in 2013. However, highest suppression of Fol inoculum was observed in amended

treatments at C:N 20 and C:N 30 (1 to 1.4 log10[CFU+1] g -1

soil) and lowest suppression in the

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non-amended control (2.5 log10[CFU+1] g -1

soil). A similar trend in Fol suppression with

significant differences was observed in 2014 with significant reduction of Fol population in

amended plots (3.2 log10[CFU+1] g -1

soil) though Fol population retrieved were higher in all

treatment than in the first year study with highest Fol population (5.7 log10[CFU+1] g -1

soil) in

non-amended control (p<0.001, Figure 3-5). The Fol populations from packets that was not

buried in soil was 6.07 log10[CFU+1] g -1

soil.

At the end of ASD termination Fusarium counts from soil samples was significantly higher in

ASD20 and lower in fumigated plots (p<0.001) in 2013. However, across all treatments in 2014,

there was no interaction and no significant differences for soil Fusarium counts (Table 3-4), but

reported low after ASD few weeks of fumigation treatment. The endemic Fusarium population

identified was F. solani, F. foetens, and F. concolor.

4. Discussion

Separate analysis was performed for pot trials as we observed variation in trials in terms of

interaction between organic amendment and C:N treatment, soil pH, cumulative anaerobic

condition, and Fusarium inoculum survival. In addition, separate analysis for field trials was

conducted due to separate fields selected for ASD study.

Soil pH reduction in ASD treatment is one of the indicators of organic acids production, such as

acetic acid and butyric acid (Momma et al., 2006; Momma et al., 2011) that is attributable to

breaking down of amendment carbon. Several studies have reported reduced pH after

amendment incubation in ASD (Katase et al., 2009; Butler et al., 2012b; Hewavitharana and

Mazzola, 2013) and in our pot studies, we also observed lowering of soil pH by 0.8 to 0.9 units

across all treatments including non-amended control; lowest soil pH was recorded at C:N 10:1.

On the contrary, significant lowering of soil pH after the ASD treatment is not necessary (Butler

et al., 2014b; Huang et al., 2016). Soil pH in 2013 was higher than initial soil pH, which was

similar to the first year field study of McCarty et al. (2014) however, the soil pH change in the

second year though negative, was not significant. Generally, not only amendment types and soil

microbial activity influences soil pH, soil types also control pH instability. In the field study, the

clay loam soil type may have a higher buffering capacity than soil collected for the pot study

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(sandy soil), which limited any pH changes. Also recorded soil pH value in the field study,

where sampling covers a large area than in the pot study was expected to have a lower pH

change than at the site of microbial activity (Strong et al., 1997; Katase et al., 2009) as low pH

contributers, i.e organic acid generated during ASD, can be readily escape once soil is exposed

(Jones et al., 2003). Studies have reported that Fusarium oxysporum survival is observed in wide

pH range (Manandhar and Bruehl, 1972) and control is observed only if pH of greater than nine

units is obtained (Jones and Woltz, 1969).

As discussed above, overall soil pH may or may not be critical factors for reduction of the

number of Fusarium propagules during ASD treatment, but reduced soil conditions due to loss of

oxygen by soil microbes and limited gas exchange inside plastic mulch may play a significant

role in the survival of Fusarium. Though our soil temperature in both studies was not high

enough to provide any lethal effect, increased accumulation of anaerobic conditions was

considered to create unfavorable soil conditions for pathogens. In our pot trials, anaerobic

conditions was higher in all of our amended pots along with control (>140,000 mV h).

Application of organic amendments as carbon source significantly increased accumulation of

anaerobic conditions (>179, 000 mV h) indicating high microbial activity. This was

approximately three- to four-fold greater than the set threshold level of 50,000 mV h that is

expected to have such effect (Shennan et al., 2010). Similarly, in our field study, iron

oxyhydroxide paint removal indicated high anaerobic condition in amended plots suggesting that

microbial activity is more prevalent in amended plots than in control. Low anaerobic condition in

plot amendment with 2 mg C g-1

soil compared to 4 mg C g-1

soil indicated that anaerobic

condition is largely affected by the rate of carbon than C:N ratios.

Significant reduction of Fol populations (44 to 87%) by ASD with high anaerobic condition was

observed when dry molasses at the rate of 4 mg C kg-1

of soil at various C:N ratios was applied

in the field plots. Surprisingly, suppression of Fol in pot trials with the same rate of carbon at

different C:N ratios was not consistent among two pot trials and the field study. Use of dry

molasses showed higher suppression of the pathogen in pot trials than wheat bran. However, lack

of clear effects of amendments as compared to control treatment in trial 2 suggests that initial

soil properties and accumulation of anaerobic condition may have influenced inoculum survival.

As mentioned above, the anaerobic condition for pot studies was high enough to suppress Fol

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inoculum at moderate soil temperature and this is attributed to an increase in anaerobic bacteria

especially Clostridia (Mowlick et al., 2013a; Mowlick et al., 2013b). We believe even in the

covered but non-amended treatments there could be similar conditions and other synergistic

effects of organic acid, volatiles like allyl alcohol and reduced form of Fe and Mn, which are

usually reported during ASD treatment (Momma et al., 2011; Hewavitharana et al., 2014) may

also exist. Though production of these toxic product for non-amended control may not reach the

suppressive threshold level, we believe that the higher anoxic condition, which is prevalent at the

greater depth may have significantly reduced Fol survival (see Table 2). Ebihara and Uematsu

(2014) also reported that anaerobic condition without any amendments can make significant

contribution in the reduction of Fusarium oxysporum propagules but higher incubation period

(>30 weeks) required for complete eradication at 30°C. In trial 2, besides the anaerobic

condition, the relationship between Fusarium survival and C:N of soil at the end of treatment

with high C:N in control may have attributed to no difference. Also, soil nutrients present in

different forms impacts Fusarium survival and disease development (Jones and Woltz, 1969;

1970; Huber and Thompson, 2007) and higher nitrate N and ammonium N in trial 2 than trial 1

explain lower Fusarium populations in control in an anaerobic condition.

Usually, saturating and covering the soil without any amendments have failed to control Fo in

field studies (Blok et al., 2000; Mowlick et al., 2012b; Mowlick et al., 2014). However, ASD is

not always effective against Fusarium suppression. For instance, similar to our pot studies,

artificial infestation of Fo in soil treated with cover crop amendments cowpea and pearl millet

(Butler 2012a) incubated for 3 weeks failed to suppress Fol. Similarly, a previous pot study on

Fusarium root rot of common bean also produced inconsistent result in disease suppression that

utilized cool-season cover crops as a C source Butler et al. (2014b). When by-products like

wheat bran at the rate of 1 kg m-2

(Horita and Kitamoto, 2015) and rice bran, or a combination of

rice bran and mustard applied as a C source (Daugovish et al., 2013), also failed to control the Fo

population under soil temperatures that ranged between 25 to 35°C indicating a high carbon

requirement for disease suppression.

In this research, although higher C rates at different C:N were maintained for organic

amendments, it is possible that different forms of organic C and their relative recalcitrance to

microbial decomposition might have influenced our results. Soil suppression and microbial

105

activity is influenced by amendment quality (Steiner and Lockwood, 1970; Senechkin et al.,

2014) and Fol suppression in soil has always been challenging (Momma et al., 2006; Huang et

al., 2015). Addition of amendments creates confounding effects in soil, i.e., amendment may

protect or even increase the Fusarium population nullifying the fungistatic property of soil

beforehand when some air is present (Zakaria and Lockwood, 1980; Kamble and Bååth, 2014;

Morauf and Steinkellner, 2015) and after anoxic condition Fol may exist by nitrate respiration

(Zhou et al., 2001); thus, preventing complete eradication of Fol. Further, instead of pathogen

incorporation in soil, use of small bags for Fol propagules in our study may facilitate the

propagules to escape the toxic effect of organic acids and other volatiles generated during ASD.

While the effect of amendments could have been different if propagules were not colonized in

rice and placed in media plates as in Hewavitharana et al. (2014), exposing propagules to

volatiles from amended pots.

Only a few studies on pathogens and nematodes have reported C:N of amendments used in the

ASD. The amendments C:N applied to suppress Fo inocula were found effective at ranges of 10

to 40 C:N ratios. Cover crops utilized in Butler et al. (2012a) ranged from 10 to 40. The lowest

ratio found for cowpea (C:N 10), highest for sorghum sudan (C:N 40) and dry molasses, pearl

millet and sunn hemp had C:N 21 to 30. Mustard seed meal, composted steer manure, grass

residues and rice bran used in Hewavitharana et al. (2014) had C:N of 10 to 20. These studies

were conducted in similar environment to the present study with sandy soil, soil temperatures not

exceeding 25°C and soil depth in pots of 5- to 15-cm depth. Only composted steer manure at

lower C:N, and cowpea and liquid molasses at higher C:N failed to suppress Fusarium

populations. In our pot trials also at higher C:N of 40 in our pot trials Fol was not suppressed and

between two amendment types wheat bran was not effective in controlling Fol at all C:N ratios.

Dry molasses showed 24 to 30% of suppression in trial 1, though no significant results were

observed in trial 2. It was not surprising to see higher Fol decreased in field conditions (<35% at

C:N 10, 36 to 58% in other C:N ratios) at higher C rate when compared with control since

pathogen inoculum is better exposed (Butler et al. 2012a) and the readily decomposable nature of

dry molasses was more effective against pathogen inoculum than in pots.

We analyzed soil samples collected from the field for endemic Fusarium populations and these

were similar across all treatments in 2014. Not all endemic Fusarium spp. are pathogens and soil

106

samples contained a mixture of other pathogens, saprophytes, and beneficial endophytes. Similar

results, without possible effect of ASD on the relative abundance of endemic Fusarium

populations have been reported in Rosskopf et al. (2014) and Huang et al. (2016). However, we

observed a negative effect of fumigation on endemic Fusarium population after application of

fumigation in field trials indicating greater chance of potential loss of other beneficial organisms.

Pepper yield data from the field study showed increased productivity in ASD treatment

compared to non-amended covered treatment and the fumigant treatment. It is not surprising to

observe higher yield from ASD treated plots than fumigated plots as addition of amendments in

soil is reported to increase microbial response to promote plant health. Our meta-analysis review

(Chapter 1) also pointed to the role of increased yield in ASD. However, ASD may have more

potential as a disease control practice in lower temperature regimes. More ASD studies with

naturally infested field soil need to be explored to allow growers’ confidence to rely on this

technique.

5. Conclusion

To summarize, dry molasses was the first amendment to be studied for potential control of

Fusarium population and it was found to be more potent than wheat bran maintained at C:N 20

to 30. Fol suppression using dry molasses with a lower C:N and a lower rate is not

recommended, which was also evident from poor bacterial pathogen suppression in study

conducted by McCarty et al. (2014). However, soil nutrient analysis after ASD treatment could

be helpful in drawing conclusive recommendations (Butler et al., 2014a). At this time, our results

can help growers decide on the amendment rate and C:N for Fusarium control.

107

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112

Appendix

Table 3-1. Gravimetric soil moisture content (g g-1

) of soil after 3 weeks of ASD treatment, pot

study

Carbony No. of observations Moisture content (g g

-1)

a

Trial 1 Trial 2

Control 8 0.23 ± 0.01 b 0.23 ± 0.01 c

DM 16 0.26 ± 0.01 a 0.24 ±0.01 b

WB 16 0.25 ± 0.01 a 0.26 ± 0.01 a

p-value <0.0001 <0.0001

C:N

CN10 8 0.24 ± 0.01 bc 0.25 ± 0.01 a

CN20 8 0.26 ± 0.01 ab 0.25 ± 0.01 ab

CN30 8 0.26 ± 0.01 a 0.26 ± 0.01 a

CN40 8 0.26 ± 0.01 ab 0.25 ± 0.01 a

Control 8 0.23 ± 0.01 c 0.23 ± 0.01 b

p-value <0.0001 .01

aNumbers represent the soil moisture mean ± standard error respective to number of observation.

Different letters represent significant differences at p<0.05 within columns for each category

according to Fisher’s protected LSD test.

yDry molasses (DM), Wheat bran(WB), C:N ratio 10:1 (10), C:N ratio 20:1 (20), C:N ratio 30:1

(30) and C:N ratio 40:1(40)

113

Table 3-2. Effect of location of Fusarium inoculum packet in the pot on Fusarium populations,

pot assays

Location

treatment Fusarium population

a

Trial 1 Trial 2

15-cm 3.06+0.17 b 3.12+0.15 b

Control, 15-cm 3.52+0.27 ab 2.27+0.57 c

5-cm 3.67+0.13 a 4.2+0.1 a

Control, 5-cm 3.67+0.31 ab 4+0.17 a

p-value <0.025 <0.001

aNumbers represent the mean soil Fusarium populations (log10[CFU+1]g

-1 inoculum) ± standard

error of four replicated pots. Different letters represent significant differences at p<0.05 within

columns for each category according to Fisher’s protected LSD test.

114

Table 3-3. Effect of C: N ratio soil treatment on soil gravimetric moisture content (g g-1

) in field condition

C:Na

Year 2013 Year 2014

Pre ASD End ASD Post ASD Pre ASD End ASD Post ASD

ASD10 2.5 ± 0.16 2.9 ± 0.05 ab 3.0 ± 0.2 2.7 ± 0.19 3.4 ± 0.24 2.5 ± 0.17

ASD20 2.6 ± 0.09 3.1 ± 0.1 a 2.9 ± 0.09 2.9 ± 0.11 2.9 ± 0.29 2.7 ± 0.31

ASD30 2.7 ± 0.07 2.8 ± 0.15 b 3.1 ± 0.15 3.1 ± 0.26 3.9 ± 0.55 2.4 ± 0.27

ASD40 2.5 ± 0.05 2.5 ± 0.17 cd 2.7 ± 0.14 3.0 ± 0.08 2.9 ± 0.35 2.9 ± 0.25

Fum 2.5 ± 0.13 1.5 ± 0.53 d 2.1 ± 0.72 2.7 ± 0.05 3.3 ± 0.33 1.8 ± 0.28

LCASD30 2.7 ± 0.10 2.9 ± 0.08 ab 2.8 ± 0.13 2.8 ± 0.11 3.1 ± 0.31 2.4 ± 0.09

UTC 2.5 ± 0.05 2.8 ± 0.03 bc 2.9 ± 0.05 2.8 ± 0.10 3.2 ± 0.29 2.4 ± 0.18

aCarbon to nitrogen (C:N) C:N ratio 10:1 (ASD10), C:N ratio 20:1 (ASD20), C:N ratio 30:1 (ASD30), C:N ratio 40:1 (ASD40), Low

carbon C:N ratio 30:1, C rate 2 mg C g-1 soil (LCASD30) Untreated, non-amended control (UTC) and Fumigated of Methyl bromide

and chloropicrin (67:33), 200 lbs acre-1

115

Table 3-4. Effect of C: N ratio soil treatment on endemic soil Fusarium populations in field assays

C:Na

Year 2013 Year 2014

Pre ASD End ASD Post ASD Pre ASD End ASD Post ASD

ASD10 2.5 ± 0.16 2.9 ± 0.05 ab 3.0 ± 0.2

2.7 ± 0.19 3.4 ± 0.24 2.5 ± 0.17

ASD20 2.6 ± 0.09 3.1 ± 0.1 a 2.9 ± 0.09 2.9 ± 0.11 2.9 ± 0.29 2.7 ± 0.31

ASD30 2.7 ± 0.07 2.8 ± 0.15 b 3.1 ± 0.15 3.1 ± 0.26 3.9 ± 0.55 2.4 ± 0.27

ASD40 2.5 ± 0.05 2.5 ± 0.17 cd 2.7 ± 0.14 3 ± 0.08 2.9 ± 0.35 2.9 ± 0.25

Fumigated 2.5 ± 0.13 1.5 ± 0.53 d 2.1 ± 0.72 2.7 ± 0.05 3.3 ± 0.33 1.8 ± 0.28

LCASD30 2.7 ± 0.1 2.9 ± 0.08 ab 2.8 ± 0.13 2.8 ± 0.11 3.1 ± 0.31 2.4 ± 0.09

UTC 2.5 ± 0.05 2.8 ± 0.03 bc 2.9 ± 0.05 2.8 ± 0.10 3.2 ± 0.29 2.4 ± 0.18

p-value 0.4648 0.0006 0.526 0.3346 0.3159 0.0806

Numbers represent the mean soil Fusarium populations (log10[CFU+1]g-1

soil) ± standard error of four replicated plots. Different

letters represent significant differences at p<0.05 within columns for each category according to Fisher’s protected LSD test.

aCarbon to nitrogen (C:N) C:N ratio 10:1 (ASD10), C:N ratio 20:1 (ASD20), C:N ratio 30:1 (ASD30), C:N ratio 40:1 (ASD40), Low

carbon C:N ratio 30:1, C rate 2 mg C g-1 soil (LCASD30) Untreated, non-amended control (UTC) and Fumigated of Methyl bromide

and chloropicrin (67:33), 200 lbs acre-1

116

Table 3-5. Growth characteristics of 8-week-old tomato plant transplanted at 5 weeks old in pots after termination of ASD treatment

(Trial 1)

Carbony Fruit weight (g)

Fruit

(no.)

Flower

(no.)

Shoot height

(cm)

Stem diameter

(mm)

Dry shoot

biomass (g)

Dry root

biomass (g)

Control 29.8 ± 6.6 b 3.6 ± 1.2 b 16.5 ± 3.2 ab 35.6 ± 1.7 ab 8.6 ± 0.3 b 7 ± 1.3 ab 0.8 ± 0.1 a

DM 65.2 ± 8.6 a 6.4 ± 3.1 a 17.2 ± 1.9 a 37.3 ± 1.6 a 9.4 ± 0.3 a 7 ± 0.4 a 0.8 ± 0.1 a

WB 48.5 ± 7.4 ab 4.4 ± 2.4 b 10.1 ± 2.3 b 30.6 ± 1.7 b 8.8 ± 0.2 b 5.7 ± 0.6 b 0.7 ± 0.1 a

p-value 0.031 0.0309 0.0234 0.0129 0.0446 0.0312 0.6301

C:N

10 86.9 ± 5.7 a 6.9 ± 0.9 a 20.1 ± 2 a 37.9 ± 2.2 a 9.9 ± 0.3 a 8.8 ± 0.4 a 1.1 ± 0.1 a

20 69 ± 10.1 ab 5.8 ± 0.7 ab 10.1 ± 3 b 33.3 ± 2.9 a 9.1 ± 0.3 ab 4.9 ± 0.5 b 0.7 ± 0.1 b

30 47.1 ± 11.8 bc 5.9 ± 1.4 ab 14.9 ± 3.3 ab 33.1 ± 3.2 a 9.1 ± 0.3 abc 6.3 ± 0.7 b 0.7 ± 0.1 b

40 24.4 ± 2.4 c 3.1 ± 0.6 c 9.5 ± 3.3 b 31.6 ± 1.8 a 8.3 ± 0.3 c 5.4 ± 0.6 b 0.6 ± 0.1 b

Control 29.8 ± 6.6 c 3.6 ± 0.4 bc 16.5 ± 3.2 ab 35.4 ± 1.7 a 8.6 ± 0.3 bc 7 ± 1.5 b 0.8 ± 0.1 b

p-value <0.0001 0.0193 0.0421 0.3051 0.006 0.0002 0.0087

Numbers represent the mean ± standard error of four replicates of tomato plant for fruit weight, fruit number, flower number, shoot

height, stem diameter, dry shoot biomass, dry root biomass. Different letters represent significant differences at p<0.05 within


yDry molasses (DM), Wheat bran(WB), C:N ratio 10:1 (10), C:N ratio 20:1 (20), C:N ratio 30:1 (30) and C:N ratio 40:1(40)

117

Table 3-6. Growth characteristics of 8-week-old tomato plant transplanted at 5 weeks old in pots after termination of ASD treatment

(Trial 2)

Carbony

Fruit weight

(g) Fruit (no.) Flower (no.)

Shoot height

(cm)

Stem diameter

(mm)

Dry shoot biomass

(g)

Dry root

biomass (g)

Control 9.5 ± 3.6 c 3.3 ± 0.6 b 19.5 ± 3.1 ab 31.9 ± 1 b 8.6 ± 0.3 b 5.6 ± 0.8 b 0.9 ± 0.1 a

DM 49 ± 5.4 a 6.4 ± 0.5 a 19.9 ± 3.5 a 36.4 ± 1.5 a 9.4 ± 0.2 a 7.1 ± 0.6 a 1 ± 0.1 a

WB 25.4 ± 4.5 b 6.4 ± 1 a 20 ± 2.8 b 32.6 ± 1.3 b 8.4 ± 0.2 b 5.4 ± 0.6 ab 0.8 ± 0.1 a

p-value <0.0001 0.011 0.9836 0.0463 0.0075 0.0496 0.2913

C:N

10 53.1 ± 6.4 a 7.4 ± 1.6 a 17.9 ± 4.3 a 34.6 ± 2.5 a 9.5 ± 0.3 a 6.6 ± 0.8 a 1 ± 0.1 a

20 28.4 ± 10 b 6.9 ± 1.1 a 22 ± 4.5 a 35.9 ± 1.1 a 8.8 ± 0.4 a 6.1 ± 1.5 a 1 ± 0.1 a

30 34.9 ± 6 b 7.4 ± 0.9 a 20.5 ± 2.7 a 35.6 ± 1.5 a 8.6 ± 0.2 a 6.4 ± 0.3 a 0.8 ± 0.1 a

40 32.3 ± 7.9 b 4 ± 0.5 b 19.5 ± 6.2 a 31.9 ± 2.8 a 8.6 ± 0.4 a 5.8 ± 0.5 a 0.8 ± 0.1 a

Control 9.5 ± 3.6 c 3.3 ± 0.6 b 19.5 ± 3.1 a 31.9 ± 1 a 8.6 ± 0.3 a 5.6 ± 0.3 a 0.9 ± 0.1 a

p-value 0.0003 0.0046 0.9854 0.2529 0.1717 0.2039 0.5686

Numbers represent the mean ± standard error of four replicates of tomato plant for fruit weight, fruit number, flower number, shoot

height, stem diameter, dry shoot biomass, dry root biomass. Different letters represent significant differences at p<0.05 within


yDry molasses (DM), Wheat bran(WB), Carbon to nitrogen (C:N) C:N ratio 10:1 (10), C:N ratio 20:1 (20), C:N ratio 30:1 (30) and

C:N ratio 40:1(40)

118

Figure 3-1. Effect of amendment C:N ratio on soil pH at treatment termination, pot study.

Bars indicated by different letters are significantly different at p<0.05 according to Fisher’s

protected LSD test. Error bars indicate standard error with four replicates. Control=non-amended

control, CN10=C:N ratio 10:1, CN20=C:N ratio 20:1, CN30=C:N ratio 30:1 and CN40=C:N

ratio 40:1, DM=dry molasses, WB=wheat bran

b*

a

a

a

b*

a a

a

4.6

4.8

5

5.2

5.4

5.6

5.8

CN10 CN20 CN30 CN40

So

il p

H

Trial 1 Dry molassesWheat branmean DMmean WBControl

bc

a ab

a

*c

a ab

a

4.6

4.8

5

5.2

5.4

5.6

5.8

CN10 CN20 CN30 CN40

. S

oil p

H

Carbon to Nitrogen ratio

Trial 2

119

Figure 3-2. Effect of amendment C:N ratio on cumulative anaerobic condition during ASD, pot

study.





*a *a *ab *bc

*cd

*c

*cd *d

10000

60000

110000

160000

210000

260000

310000

360000

CN10 CN20 CN30 CN40

Cu

mu

lati

ve

an

ae

rob

icit

y (

mV

hr)


*a *a *ab

*bc

*abc

*ab

*c

*abc

10000

60000

110000

160000

210000

260000

310000

360000

CN10 CN20 CN30 CN40

Cu

mu

lati

ve

an

ae

rob

icit

y (

mV

hr)


Trial 2

120

Figure 3-3. Effect of amendment and amendment C:N ratio on Fusarium populations during

ASD treatment, pot study.

Bars indicated by different letters are significantly different, p<0.05 according to Fisher’s




*c *c

*c

a ab

ab b ab

0

1

2

3

4

5

6

CN10 CN20 CN30 CN40

Fu

sa

riu

m o

xy

sp

oru

m

(lo

g1

0[C

FU±1]

g-1

so

il)


0

1

2

3

4

5

6

CN10 CN20 CN30 CN40

Fu

sa

riu

m o

xy

sp

oru

m

(lo

g1

0[C

FU

+1

] g

-1 s

oil)


Trial 2

121

Figure 3-4. Effect of amendment C:N ratio on percentage of iron oxyhydroxide paint removal

following ASD treatment, field study, 2013-2014.


protected LSD test. ASD10=C:N ratio 10:1, ASD20=C:N ratio 20:1, ASD30=C:N ratio 30:1,

ASD40=C:N ratio 40:1, LCASD30=C:N ratio 30:1, C rate 2 mg C g-1 soil (LC ± ‘low carbon’)

and UTC=Untreated, non-amended control

a

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Carbon to nitrogen ratio

Year 2013

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CeH 2014

122

Figure 3-5. Effect of amendment C:N ratio on Fusarium oxysporum inoculum populations

following ASD treatment, field study, 2013-2014.


protected LSD test. The dashed line represents Fo populations (6.07 log10[CFU+1]g-1

soil) from

packets not buried in the field. Bars indicated by different letters are significantly different,

p<0.05 according to Fisher’s protected LSD test. ASD10=C:N ratio 10:1, ASD20=C:N ratio

20:1, ASD30=C:N ratio 30:1, ASD40=C:N ratio 40:1, LCASD30=C:N ratio 30:1, C rate 2 mg C

g-1 soil (LC ± ‘low carbon’) and UTC=Untreated, non-amended control

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Carbon to nitrogen ratio

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Unburied bag

123

Figure 3-6. Mean pepper fruit numbers ha-1

harvested for the year 2013 and 2014. i. total fruit, ii.

marketable fruit (Fancy+US No.1 + US No.2), iii. Fancy and iv. culled fruit

Within yield class and year, means indicated by different letters are significantly different, Tukey

test, p<0.05. Error bars represent std. error at n=4.

124

Figure 3-7. Mean pepper yield (mt ha-1

) for the year 2013 and 2014. i. total yield, ii. marketable

yield (Fancy + US No.1 + US No.2), iii. Fancy and iv. culled yield.

Within yield class and year, means indicated by different letters are significantly different, Tukey

test, p<0.05. Error bars represent std. error at n=4.

125

Chapter 4

Effects of amendment C:N ratio and carbon rate on germination

and parasitism of sclerotia of Sclerotium rolfsii following anaerobic

soil disinfestation

126

A version of this chapter is a manuscript in preparation for Plant Disease by Utsala Shrestha,

Bonnie H. Ownley and David M. Butler.


analysis, results interpretation and writing.

127

Abstract

Growth chamber and field studies were carried out with dry molasses and/or wheat bran

maintained at different C:N ratios (10:1, 20:1, 30:1 and 40:1) to evaluate the ASD effect on

mortality and parasitism of Sclerotium rolfsii. A growth chamber study with dry molasses

amendment at rates of 2, 4, 6, and 8 mg C g-1

soil (C:N ratio 30:1) was also carried out with

sclerotia of S. rolfsii to examine the optimum ASD amendment rate. Two polyethylene mesh

bags with 10 sclerotia each were buried at 5- to 10-cm depths in amended field soil and in pots

containing field soil and sand. Sclerotial germination and parasitism were accessed after three

weeks of ASD treatment. In the pot study, there were no significant interactions among carbon

amendment, C:N ratio and soil depth observed for sclerotial germination, decomposition and

parasitism, while significant relationships were observed between sclerotial germination and

parasitism by Trichoderma treatments. Sclerotial germination was significantly reduced in all

amended pots regardless of C:N ratios (0.6-8.5%) and carbon rates (7.5-46%) as compared to

non-amended controls (21-36% and 61-96%, respectively). In the field study, sclerotial

germination was significantly reduced to 50% and ranged from 0.8-11%. Similarly, during ASD

treatment, amendments had a significant positive impact on colonization of sclerotia by

Trichoderma spp., with higher parasitism in all C:N ratio treatments, and at 2 or 4 mg C g-1

soil

(>80%) compared to the control. In the field study, sclerotial parasitism by Trichoderma spp.

was predominant, however, other mycoparasites, i.e., Aspergillus spp., Fusarium spp., Mucor

spp., and other fungi were present. Our results suggest that ASD with carbon amendment

application at the rate of at least 4 mg C g-1

soil induces optimum anaerobic soil conditions,

facilitating suppression of sclerotia of S. rolfsii and enhances parasitism by beneficial fungi

including Trichoderma spp.

Keywords: Anaerobic, amendments, C:N ratio, carbon rate, parasitism, Sclerotium rolfsii,

Trichoderma

128

1. Introduction

With the mandated phase out of the ozone depleting fumigant, methyl bromide (MeBr),

vegetables and small fruit growers are seeking for an alternative method to control plant

diseases, pests, and weeds in their production system. This has led to the introduction of several

alternatives to MeBr for high value crops. However, these alternatives have not met the technical

superiority and satisfaction of the growers compared to the use of MeBr fumigation (Belova et

al., 2013). In addition, these alternatives also face regulatory restrictions and limitations (Martin,

2003) because of one or more constraints like geographic limitations, lack of efficacy, human

safety concerns, and accumulation of phytotoxic materials (Csinos et al., 2002). In this scenario,

anaerobic soil disinfestation (ASD) has potential as a non-chemical alternatives to MeBr (Butler

et al., 2012b;Shennan et al., 2014; Rosskopf et al., 2015). Initiated in Japan as biological

disinfestation and as anaerobic disinfestation in the Netherland sixteen years ago, ASD has

evolved as one of the effective measures to control soilborne pathogens and nematodes.

Application of organic amendments in soil as a disease suppressor has been widely studied

(Bonanomi et al., 2010; Bonanomi et al., 2013) but using this technique to control soilborne

pathogens has increased after significant disease suppression was observed with ASD (Blok et

al., 2000; Shinmura 2004). ASD emphasizes using easily available organic amendments (OAs)

as a source of labile carbon (C) in saturated and covered soil to create anaerobic conditions,

enhance biocontrol agents, and release organic acids and volatiles as toxic compounds to

soilborne pathogens. Thus far, ASD effectiveness has been shown against soilborne fungal

pathogens. There are few studies on oomycetes and bacterial pathogens.

Sclerotium rolfsii Saccardo [Teleomorph: Athelia rolfsii (Curzi)] is a sclerotia forming

necrotrophic soilborne pathogen known to cause seedling damping off, root rot or stem rot. The

disease is commonly called southern blight. Sclerotium rolfsii is economically important as it is

cosmopolitan and infects on more than 500 plant species. In USA, S. rolfsii is reported mainly in

the southern region with tropical and sub-tropical areas and a warm temperate climate (Punja,

1985; Xu et al., 2009; Gao et al., 2015; Mehta et al., 2015); however, S. rolfsii is also an active

saprophyte surviving two to three years (Aycock, 1966) in a wide range of soil temperature and

129

soil pH (Coley-Smith et al., 1974). The fungus survives as sclerotia and can resist microbial

attack (Smith et al., 2015).

The pathogen was reported to cause stem rot in peanut as early as 1919 in Tennessee (USDA,

1919), and to date the infection from sclerotia of S. rolfsii is reported to cause huge losses in

specialty vegetables and small fruits (U. Shrestha, A.L. Wszelaki, and D.M. Butler, 2014, a UT

Extension Publication). After the phase-out of MeBr fumigation, chemical control practices offer

alternative options to control S. rolfsii. However, a gradual shift of consumer preferences to

organic food and USDA increasing their financial support to organic growers, and better pricing

of organic products (~$39 billion) in the US retail market (Herrick, 2016) have encouraged

farmers towards organic farming. In Tennessee, more than 50% of organic food consumers

wanted to increase their organic food option (Bhavsar et al., 2016) indicating increased

opportunities for organic growers. However, management of diseases such as southern blight can

be great concern to these growers including Tennessee local and non-chemical growers if other

alternatives to conventional chemicals are not available.

Alternative options such as solarization are not feasible in Tennessee due to insufficiency of

lethal temperature regime during the growing season. Although, biofumigation using brassica

plants can be a feasible alternative to inhibit mycelial growth, the inhibition of sclerotial

germination using crucifers and allyl isothiocynate seems to be cost-ineffective (Harvey et al.,

2002; Reddy, 2012). As mentioned above, ASD can be a cost effective option for Tennessee and

southeastern growers to control sclerotia of S. rolfsii as it utilizes locally available agriculture by-

products in a plastic culture system. Most of the studies showed that using various types of

amendments in ASD has reduced the viability of many sclerotial pathogens. Various

amendments, ranging from agricultural by products, cereal brans, grasses, cruciferous crops to

animal and poultry manures were incorporated in soil to test ASD effectiveness on

sclerotial/microsclerotial germination (Rosskopf et al., 2015; Strauss and Kluepfel, 2015) of

Verticilium dahlia (Blok et al., 2000; Thaning and Gerhardson, 2001; Goud et al., 2004; Shennan

et al., 2007; Shennan et al., 2009), Macrophomina (Rosskopf et al., 2010; Rosskopf et al., 2014),

and Rhizoctonia solani (Blok et al., 2000; Hewavitharana and Mazzola, 2013; McCarty et al.,

2014). All of these pathogens were well suppressed by ASD treatment. Only a few studies on

ASD technique with warm season cover crops in Florida (Butler et al., 2012b) and cool season

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cover crop in Tennessee (McCarty, 2012) have been carried out in pots to see sclerotial

germination. Reduction in sclerotial germination was reported in Florida trials with liquid

molasses; however, the result was inconsistent between trials (Butler et al., 2012b). Similarly, in

Tennessee trials only cereal rye showed a consistent reduction in sclerotial germination in both

trials indicating that the forms of carbon supplements may have an effect on the sclerotial

germination (McCarty, 2012). Thus, it is imperative to identify suitable carbon amendments and

rate to control S. rolfsii because amendments play a critical role in determining suitable

microbial population structure, decomposition rates (Akhtar and Malik, 2000), plant growth

(Rodriguez-Kabana et al., 1987) and collectively the effectiveness of ASD (Butler et al., 2012b).

In this study, we tested efficacy of ASD using organic amendments at different rates and

different carbon to nitrogen ratios (C:N) on the survival of sclerotia of S. rolfsii. In particular, our

aims were: (i) to evaluate redox potential of soils treated with carbon amendments for ASD

treatment, (ii) to examine suppression and parasitism of introduced sclerotia of S. rolfsii using

dry molasses and wheat bran at different C:N ratios, and (iii) to examine suppression and

parasitism of introduced sclerotia of S. rolfsii using dry molasses at different C rates (2 to 8 mg

Cg-1

of soil ) in growth chamber. We also examined the application of dry molasses at different

C:N ratios and rates in field conditions.


2.1 Sclerotia production and inoculum preparation

Sclerotium rolfsii was isolated from hybrid field tomatoes grown at the UT East Tennessee

Research and Education Center, Knoxville, TN, in 2005. Sclerotia were grown in petri dishes

(100 mm x 15 mm) with full-strength potato dextrose agar (PDA) for 7 days, and plugs were

transferred to the center of freshly prepared PDA plates. Cultures were allowed to grow in the

dark for 7 days at room temperature or until the mycelium grew to the edge of the petri dish.

Cultures were placed in a cold room at 3.3°C for 4 hour, and then returned to room temperature

and kept in the dark and cultures were allowed to dry to encourage formation of sclerotia. After 4

to 6 weeks, matured sclerotia were harvested, dried overnight under a laminar flow hood, and

stored in a sterile glass vial until use in the experiment. Before setting up the growth chamber

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study, ten sclerotia were sealed in a 5-cm × 5-cm permeable packet (aperture Delnet® polyolefin

fabric, DelStar Technologies, Austin, TX) and string was attached for easy retrieval from soil.

2.2 Growth chamber study

2.2.1 Amendment and C:N ratio effect

Soil (Dewey silt loam) was collected from a field at the UT Organic Crops Unit, Knoxville, TN,

sieved through 10 mm aluminum metal mesh and mixed with fine sand at a 1:1 proportion. The

basic properties of the air-dried soil mixture, such as soil pH, total C, total N, and total P, were

recorded. The soil and sand mixture were placed in tall pots (12-cm diameter and 23-cm height,

2.5 l) with two pathogen packets at 5-cm and 15-cm depths. Soil treatments consisted of two

main treatments i) dry molasses and ii) wheat bran amended with four C:N ratios 10:1, 20:1,

30:1, and 40:1; see Table 1). Control pots without any amendments were included for each main

treatment. All the pots were saturated with tap water, covered with polyethylene, and secured

with a heavy-duty rubber band. The pots were randomly positioned on the wire rack in the

environmental growth chamber with relative humidity maintained at 50%, and temperature at

25°C during day and 15°C at night. The study was completely randomized design with four

replications. Trials were carried out on 30 April and repeated on 22 May 2013.

2.2.2 Amendment C rate effect

To determine the effect of C rates on sclerotial germination and parasitism, a growth chamber

study was conducted as previously described above on June 2014 and May 2015. The treatments

included different carbon rates of dry molasses mixed with corn starch to maintain a C:N ratio of

30:1 (Table 4-1). Two non-amended controls, with or without sclerotial packets were included

also. After 3 weeks of ASD incubation, two sclerotial packets were removed from each pot to

examine sclerotial germination and parasitism, and 50 loose sclerotia were mixed within the top

2-cm of pot soil before treatment initiation to access disease pressure on tomato plant for which

six-week-old tomato seedlings (‘Florida Lanai’) planted in each pot. The design was a

completely randomized with four replications.

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2.3 Field study

Two packets of sclerotia were inserted in soil at 10-cm depth in the field established at the UT

Plateau Research and Education Center in Crossville, TN, during the second week of May in

2013 and 2014, to assess the effect of ASD on the sclerotial germination and parasitism. The soil

is classified in the Lily series (Fine-loamy, siliceous, semiactive, mesic Typic Hapludult). The

detail experimental layout and treatment application is described in Chapter 3. In short, our

experimental design was randomized complete block design with four replications and

treatments are listed in Chapter 3.

2.4 Assessment of sclerotia following ASD treatment

After ASD treatment (3 weeks), packets were collected from the pots and fields and were stored

in sealed bag (Ziploc®) at 4°C until examined. Packets containing sclerotia were washed with

tap water to remove adhering soil, sonicated for 1 min, and then surface-sterilized in 10%

commercial bleach for 1 min, followed by 1 minute in 10% ethanol. Sclerotia were carefully

removed from packets and plated onto PDA amended with 6.9 mg fenpropathrin/liter (Danitol

2.4 EC, Valent Chemical, Walnut Creek, CA) and 10 mg/liter rifampicin (Sigma-Aldrich, St.

Louis, MO), and incubated at room temperature for 4 to 6 weeks. Germination of sclerotia was

confirmed with mycelial growth and production of new sclerotia. Parasitism by Trichoderma

spp. and other microbial antagonists were observed simultaneously.

2.5 Measuring soil moisture, temperature, pH, and anaerobicity

Each pot in the growth chamber study was equipped with oxidation-reduction electrodes

(Combination ORP Electrode, Sensorex Corp., Garden Grove, CA, USA) at a15-cm depth to

measure cumulative redox potential. The temperature of the soil mixture of each representative

treated pots (two replicates) were recorded using temperature-moisture sensors at 10-cm (5TM

Soil Moisture Probe, Decagon Devices, Pullman, WA, USA). Soil samples (0- to 10-cm depth)

from each pot were collected for gravimetric soil moisture content and air-dried soil were used to

determine soil pH, which was done using 0.01 M CaCl2 buffer using a pH electrode (Orion 3-

Star Plus pH Benchtop Meter, Thermo Scientific, Waltham, MA, USA). The soil pH taken in

CaCl2 was later adjusted to soil water pH by adding 0.6 units. The cumulative anaerobic

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condition of soil over a three-week treatment period was calculated as described in Butler et al.

(2012a). In field trials, anaerobic condition was determined by iron oxihydroxide coated IRIS

tubes. Tubes were inserted in each plot at a 5- to 15-cm depth before irrigation and were

retrieved after the three-week treatment, cleaned with tap water and removal of paint was

assessed as described by Rabenhorst (2012). Ten soil cores from 5- to 15-cm depth were

collected and composited from each plot for soil moisture and soil pH.

2.6 Data analysis

Data were analyzed with Mixed Model Analysis of Variance (MMAOV) macro program in SAS

9.4 (SAS Institute, Cary, NC) and differences between means were determined using Tukey's

test (p<0.05). In repeated trials in the growth chamber study, carbon and C:N ratio effects were

analyzed separately as to completely randomized factorial analysis designs, where C

amendments, C:N ratios and depth were treated as fixed factors. Data were analyzed separately

by C amendment and depth to compare treatments with untreated control.

The growth chamber study with carbon rate was a completely randomized design with carbon

rate as fixed factor, and field study was a randomized complete block design. The data were

checked for outliers before analysis and non-normal and unequal variances were transformed

using arcsinsqrt or rank transformations specified (DAWG 2004). Untransformed means and

standard error of the mean were reported. Relationships between soil moisture, cumulative

anaerobic condition and sclerotia germination and parasitism were assessed with correlation

analysis at p<0.05.

3. Results

3.1 Growth chamber examination of ASD amendment and C:N ratio effect

A significant negative moderate correlation between sclerotial germination and Ceh (-0.4,

p<0.002) was observed in Pot assay 1, while only a weak relationship between soil pH, and Ceh

and gravimetric soil moisture was observed in pot assay 2 with correlation value of 0.3 (p<0.01).

No interaction among carbon amendment, C:N, and soil depth were observed for sclerotial

germination, decomposition, and parasitism in both trials.

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3.1.1 Soil characteristics and anaerobic conditions

Mean soil temperatures did not differ across treatment and ranged from 28.4 to 29.4°C during the

day and 18.4 to 19.3°C during the night. The volumetric water content of soil was higher in

control pots and lowest in wheat bran treated pots (data not shown). In contrast, gravimetric soil

moisture content was lowest in controls in pot assay 2 (Table 4-2). Gravimetric soil moisture

content was lower in pot assay 1 than pot assay 2. Soil pH after the anaerobic condition was

attained and did not differ across carbon amendment, but differed across C:N ratio. The lowest

soil pH was recorded in C:N10 (4.9 and 5.3, pot assay 1 and pot assay 2, respectively, Figure 4-

1A). The interaction between amendment and C:N ratio was observed for soil pH but was only

significant in pot assay 1 (p<0.04) with high pH for dry molasses treated pots. Soil pH increased

as C:N ratio increased from C:N10 to C:N30 for dry molasses. Mean cumulative anaerobic

condition did not differ among carbon treatments in both assays, but was significantly different

from untreated non-amended pots with the lowest value of 62.48 V h (Figure 4-1B). No

interaction was observed between amendments and C:N ratio and the highest mean anaerobic

condition for both assays were recorded for dry molasses (191V h) and C:N10 (210 V h).

3.1.2 Sclerotial germination and parasitism

Carbon amendments reduced sclerotial germination compared to untreated controls in both

assays (p<0.001). In pot assay 1, wheat bran amendment gave the highest sclerotial mortality

(Figure 4-3A). Both C amendments had a significant positive impact on colonization of sclerotia

by Trichoderma spp., with higher parasitism in all C:N ratio treatments (93 to 95% in pot assay 1

and 80 to 84% in pot assay 2) compared to controls (80.8 and 54% respectively, Table 4-2A).

Parasitism by other mycoparasites Fusarium and other unidentified fungi was observed, but were

not different among carbon treatments (Table 4-3) and C:N ratios, except for unidentified fungi

in pot assay 1 (Figure 4-2). When percentage sclerotial parasitism by Trichoderma was assessed

for pathogen packets retrieved from soil depths of 5-cm (top) and 15-cm (bottom), parasitism by

Trichoderma was greater at deeper soil depth (97%) in all treatments than from lower depth

(88%) in pot assay 2 (Figure 4-3B). In contrast, in the same study greater percentage of

Fusarium and other fungi was determined for sclerotia retrieved from the 5-cm depth (Figure 4-

3C, D). Among the treatments, sclerotial germination was lowest for carbon amendments at 5-

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and 15-cm depths compared to untreated controls in pot assay 2, while for dry molasses sclerotial

germination did not differ significantly from controls in pot assay 1 (Figure 4-3A). Colonization

of Trichoderma was highest at the 5-cm depth for pot assay 2 for dry molasses (95%) and wheat

bran (97%), and in pot assay 1, colonization ranged from 90 to 98% for both carbon treatments at

both depths (Figure 4-3B). Sclerotial parasitism by Fusarium was the highest for wheat bran at

15-cm depth (Figure 4-3C). Sclerotial decomposition was not significantly affected by carbon,

C:N ratio or depth of soil (Figure 4-1D).

3.2 Growth chamber examination of ASD amendment C rate

There was a significant moderate negative correlation of anaerobic condition on sclerotial

germination and a positive correlation on Trichoderma parasitism (±0.6, p<0.0001) in both

carbon rate studies. Only Trichoderma parasitism was negatively correlated with sclerotial

germination ranging from 0.6 to 0.7 at p<0.005. Mucor had a negative, moderate correlation

with sclerotial germination in both trials and positive correlation with Trichoderma only in trial

2. Fusarium and bacterial parasitism on sclerotia showed moderate positive correlation (0.4-0.5,

p<0.05).The data for the two depths were pooled as no difference was observed for the depth of

sclerotial packets in the soil.

3.2.1 Soil characteristics and anaerobic conditions

Average volumetric soil moisture in trial 1 ranged from 0.27 to 0.28 cm3 cm

-3, and in trial 2, soil

moisture was 0.23-0.27-cm3-cm

-3 in covered pots. Uncovered pots had 0.01 cm

3 cm

-3. Mean soil

temperature did not differ among carbon treatments during the treatment period, ranging from

21.3 to 26.2°C for trial 1, and 19.9 to 22°C for trial 2. Average volumetric soil moisture content

differed among treatments in both trials ranging from 0.2 to 0.3 g g-1

. Gravimetric soil moisture

content was not recorded for this study. We did not see any effect of depth of sclerotial burial on

sclerotial germination, or parasitism by Trichoderma for this study. No differences were

observed among carbon rate treatments for soil pH (Figure 4-4A). The cumulative anaerobic

condition was not affected by the amendment carbon rates of 2 to 8 mg kg-1

; however; it was

significantly higher (141 to158 V h in trial 1 and 107-150 V h in trial 2) than anaerobic condition

in the covered irrigated control and the uncovered irrigated control pots (0 to18 V h, Figure 4-

136

4B). Soil pH in trial 2 was relatively higher than trial 1 but none of the treatments differed in soil

pH value in both studies.

3.2.2 Sclerotial germination and parasitism

All ASD conditions, especially with carbon supplement, were effective in suppressing sclerotial

germination compared to the control. However, sclerotial germination over two studies showed

contrasting results. In trial 1, higher carbon level (>2 mg C g-1 of soil) was the most effective in

reducing sclerotial germination averaging 7.5% in C4 to 13.9% in C8. While in trial 2, carbon

rates at 8 mg C g-1 of soil or lower were more effective in repressing sclerotial germination

(Figure 4-4C).

Sclerotial parasitism by Trichoderma was higher at all levels of carbon amendment compared to

the controls in both trials (Figure 4-4D). However, in trial 1, the anaerobic condition

supplemented with any level of carbon amendments was more effective for parasitism of

sclerotia (54 to 98%) compared to controls. In the anaerobic condition without carbon

supplement, in trial 2 there were no significant differences between the anaerobic condition

without carbon supplement and the anaerobic condition supplemented with a higher carbon

source (82%, C6). Nevertheless, anaerobic conditions supplemented with a lower level of carbon

(89% with C6 to 98% with C4) were significantly more effective in parasitism of sclerotia

suggesting that a higher carbon rate (<6 mg C g-1

soil) is not required to control sclerotia.

Sclerotial parasitism by bacteria and Mucor were observed in all carbon treatments with the

highest percentage of parasitism recorded for C6 (32% and 14%, respectively in trial 1) and C8

(25% and 15%, respectively in trial 2). Fusarium colonization in both trials and other

unidentified fungi in trial 1 were observed (Figure 4-5). This indicates that besides Trichoderma,

other diverse microbes parasitize sclerotia, especially when carbon rates are higher.

After termination of the ASD treatment in growth chamber studies, pots were transferred to the

greenhouse where 5-week-old tomato seedling were transplanted into potting mix and wilting

and stem colonization were examined each week. Although, some yellowing and dry rot leaf

symptoms of early blight (Alternaria solani) were observed in both trials, occurrence of southern

137

stem blight was not observed. The mean fruit number was significantly higher for pots amended

with carbon rate 4 mg C g-1

of soil (Table 4-4).

3.3 Field examination of ASD effect on anaerobic conditions, sclerotial germination and

parasitism

In the field studies, no significant correlations of sclerotial germination with soil moisture, soil

pH, and parasitism by mycoparasites were observed in trial 1. However, there was a moderate

negative relationship of Trichoderma parasitism and IRIS tube paint removal (p<0.04) and

positive relationship with soil moisture, initial soil pH, other fungi and Aspergillus parasitism of

sclerotia in 2013 (Pearson correlation = 0.4, p<0.04). In year 2014, sclerotial germination had a

moderate negative correlation with Trichoderma parasitism of sclerotia (Spearman correlation =

0.7, p<0.001). In addition, soil pH and IRIS tube paint removal was moderately correlated

(Spearman correlation = 0.4, p<0.01).

Average soil temperatures did not differ among ASD treatments in either field study. In field

assay 1, mean soil temperatures ranged from 22 to 25°C and in field assay 2, slightly higher

mean soil temperatures were observed with a range between 24°C and 25°C. Before treatment

application, gravimetric soil moisture ranged between 2.5 to 2.7 g g-1

and slightly higher

gravimetric soil moisture were recorded in field assay 2, ranging from 2.7 to 3.1 g g-1

(Table 4-

5). Soil moisture content in field assay 1 after a three-week incubation period was significantly

high in ASD20 (3.1 g g-1

) while high gravimetric soil moisture ranged from 2.9 to 3.9 g g-1

. No

decline in soil pH among treatments was observed in both studies. Cumulative anaerobic

condition for field indicated by the IRIS paint removal percentage study is reported in Chapter 2

(Figure 3-4). The maximum paint removal in carbon treated pots with 4 mg C g-1

of soil

regardless of C:N ratio with 31 to 42% in 2013, and 23 to 33% in 2014. These results suggest

enhanced anaerobic condition. Lower anaerobic activity was observed for ASD treatments, with

a carbon rate of 2 mg C g-1

soil (6 to 28%) compared with the untreated control (1 to 16%).

In 2013, sclerotial germination was relatively low among C-amended treatments, ranging from

1% in ASD40 to 11% in ASD10 and 20, compared to the untreated control (27%), but this

difference was not significant (p=0.06). However, in the 2014 study, the germination of sclerotia

138

was significantly low in all carbon treated plots (2 to 31%) as compared to control (31%, Figure

4-6A). This is attributed to higher sclerotial parasitism by Trichoderma spp., Fusarium spp.,

Mucor and other fungi in all carbon amended pots (Figure 4-6B, C, D). We also observed

Aspergillus parasitism of sclerotia in 2013 and bacterial parasitism in 2014. Surprisingly, the

results were not significant in both studies (p=0.07 for Trichoderma and p>0.1 for all fungal

parasitism) except for Mucor in 2014 (p<0.02).

4. Discussion

Results from various studies have provided strong evidence that ASD is effective for control of

various soilborne pathogens (see Chapter 1). Application of appropriate amendments at

appropriate rates is crucial to increase ASD effectiveness. Amendments and soil C:N ratios

always impact microbial populations (Shaban et al., 1998; Akhtar and Malik, 2000), and plant

growth (Rodriguez-Kabana et al., 1987). Our previous study showed that ASD with different

amendment C:N ratios was effective for control of yellow nutsedge (see Chapter 2). More

specifically, a C:N ratio of 10:1 was found to be the best for yellow nutsedge tuber germination

and production. In this study, similar to yellow nutsedge and the Fusarium study, we tested the

effectiveness of ASD using dry molasses or wheat bran as a carbon source that had been adjusted

with soybean meal or corn starch to form different C:N ratios. In parallel with our previous

studies, this study also provided evidence that ASD is effective in controlling S. rolfsii.

Effectiveness for control of sclerotia was measured in terms of sclerotial germination and

sclerotial parasitism by various soil microorganisms. The majority of sclerotia recovered from

ASD treatments failed to germinate. Among the sclerotia germinated, most of sclerotia were

incapacitated by mycoparasites and only a few germinated without any parasitism. The presence

of endemic Trichoderma, Fusarium, and other fungi as mycoparasites of sclerotia revealed that

sclerotia were either weaken or lysed post-ASD treatment. Sclerotial parasitism by Trichoderma

was abundant in all studies; however, we also observed Aspergillus parasitism in field assay 1

and bacterial parasitism in field assay 2. Trichoderma spp. are the most studied mycoparasites

that are known to produce bioactive metabolites (Ownley et al., 2009), and produce enzymes like

β-1,3-glucanase and chitinase as a means to degrade sclerotial protective outer layer (Elad et al.,

1984) or reduce of hyphal growth (Madi et al., 1997). It has been reported that eruptive

139

germination of sclerotia in soil or breaking of the rind due to treatment effect can release

nutrients that stimulate the colonization of sclerotia (Smith, 1972). We also observed

zygomycetes (Rhizopus, Mucor) as sclerotial parasites from the field soil that might have

attributed more nitrate accumulation in the soil. The presence of Mucor is supposed to cause

ammonium toxicity to sclerotia in soil. However, we did not observe any significant suppression

of sclerotia by other mycoparasites as compared to the control treatment. In addition, it is also

expected that sclerotia can be targeted by anaerobic bacteria especially Clostridia (Adandonon et

al., 2015); however, our experiment did not test sclerotial decomposition by anaerobic bacteria.

With the application of amendments, regardless of C:N ratio and C rates, high cumulative

anaerobic conditions were recorded within 3 weeks of ASD treatment, which was similar to our

previous studies. Consistent relationships among anaerobic conditions, sclerotial germination,

and Trichoderma parasitism of sclerotia was observed only in the growth chamber studies with

carbon rate effect. In the pot study, C:N ratio10:1 had the lowest soil pH, which was similar to

previous pot studies conducted for nutsedge and Fusarium. We also found that in ASD

treatments, endemic Trichoderma parasitized higher number of sclerotia compared to the control.

However, under field condition, sclerotial colonization by Trichoderma was lower than in pot

studies and not significant. This could be due to diverse microorganism present in field soil that

could lead to more competition for parasitism. In addition, we did not observe significant

differences among different C:N ratios to repress sclerotial germination or sclerotial parasitism

by Trichoderma in both pot and field conditions, although Trichoderma populations are reported

to increase in soil with high soil C:N ratio (40:1) when amendment with glucose or cellulose is

applied (Shaban et al., 1998). Overall, our growth chamber studies showed that ASD was

effective in reducing sclerotial germination.

We observed that a carbon rate of 4 mg C g-1

soil was the optimal amount of carbon to suppress

sclerotial germination. Likewise, using 2 or 4 mg C g-1

soil with ASD treatment resulted in the

highest sclerotial parasitism by Trichoderma. Overall, these results suggest that 4 mg C g-1

soil

was the optimum amount of carbon to be used with ASD for sclerotial mortality. Further, studies

with 4 mg C g-1

soil with different amount of nitrogen to adjust different C:N ratio also showed

higher mortality of sclerotia.

140

Studies have suggested that increased depth of sclerotial burial increased the mortality of the

sclerotia (Imolehin and Grogan, 1980; Smith et al., 1989) as increased in soil pressure resulted in

sclerotial substrate leakage (Punja et al., 1984) and presence of soil moisture further compacted

the soil during ASD. In our study, the effectiveness of carbon supplements at different C:N ratio

to suppress sclerotia was not effective at deeper depth (15-cm) in pot assay 1 which may be

attributable to low moisture (Table 4-2) as sclerotia are susceptible to exposure to excess

moisture for more than 50 days (Moore, 1949; Abawi et al., 1985). Overall, ASD treatment using

both dry molasses and wheat bran as carbon amendments was more effective in inducing

sclerotial mortality. In the carbon rate study with dry molasses, we observed higher sclerotial

germination in trial 2, compared to trial 1.This could be due to the use of fresher sclerotia in trial

2 that were not as dry as sclerotia that were used in trial 1 and Smith et al. (1989) reported that

dried sclerotia may have reduced longevity. Besides the trial 2 carbon rate study, we used

sclerotia produced on PDA in our experiments, rather than sclerotia produced in soil which

confirms the presence of parasites after ASD treatment were solely from soil in which they were

buried.

5. Conclusion

In short, our results showed that ASD can effectively reduce sclerotial germination percentage.

With ASD treatments, the majority of sclerotia failed to germinate, and if they germinate,

Trichoderma in most of the cases parasitized those sclerotia and in some instances, other fungi

parasitized sclerotia. We observed that this phenomenon was more efficient at lower soil depth

(5-cm) than higher soil depth (15-cm). We also observed that 4 mg C g-1

soil was the optimal

amount of carbon amendments to add. However, as we presumed, we did not observe any

difference in the effect of different C:N ratios on sclerotial mortality. Thus, our results support

the notion that ASD can be one of the alternatives to control S. rolfsii. Addition of dry molasses

or wheat bran as organic amendments not only reduce pathogen inoculum, but also contribute to

increased population of beneficial organisms that suppress S. rolfsii in the soil (Beute and

Rodriguez-Kabana, 1981). Moreover, organic matter enrichment obtained with addition of

amendments, which is important to ameliorate soil structure and soil fertility (Bulluck et al.,

2002; Bailey and Lazarovits, 2003; Blum and Rodríguez-Kábana, 2004).

141

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146

Appendix

Table 4-1. Treatment: carbon to nitrogen ratio, carbon amendments, and amendments rates

Treatments label C:N ratio Carbon rate

Rate of amendments

Wheat

bran

Soybean

meal

Corn

starch

Carbon:Nitrogen pot

study mg C g

-1 soil g kg

-1 of soil

C:N10 10:1 4 7.8 1.8 -

C:N20 20:1 4 6.4 - 3.3

C:N30 30:1 4 4.2 - 5.6

C:N40 40:1 4 3.2 - 6.7

Dry

molasses

Soybean

meal

Corn

starch

C:N10 10:1 4 6.4 3.6 -

C:N20 20:1 4 9.4 0.9 -

C:N30 30:1 4 10.3 - 0.1

C:N40 40:1 4 7.7 - 2.6

Carbon rate pot study

ASD2 30:1 2 5.1 - 0.04

ASD4 30:1 4 10.3 - 0.09

ASD6 30:1 6 15.4 - 0.13

ASD8 30:1 8 20.5 - 0.17

Field Study kg m

-2

ASD10 10:1 4 0.86 0.48 -

ASD20 20:1 4 1.26 0.12 -

ASD30 30:1 4 1.38 0.01 -

ASD40 40:1 4 1.03 0.35 -

lowASD 30 30:1 2 0.69 0.01 -

147

Table 4-2. Mean gravimetric soil moisture, soil pH and anaerobic condition during treatment and

post treatment from C:N ratio pot assay conducted in the growth chamber

Carbon treatments Gravimetric soil

moisture Soil pH

Soil anaerobic

condition

g g-1

units V h

Pot assay 1

CTRL 0.22 ± 0 6 ± 0 42.7 ± 15.3 b

Dry molasses 0.22 ± 0 5.9 ± 0.1 185.3 ± 10.6 a

Wheat bran 0.22 ± 0 5.8 ± 0.1 157.6 ± 12.8 a

p value 0.607 0.4923 <0.0001

Pot assay 2

CTRL 0.24 ± 0 b 5.3 ± 0 82.3 ± 20.3 b

Dry molasses 0.27 ± 0 a 5.4 ± 0.1 196 ± 10.4 a

Wheat bran 0.26 ± 0 a 5.3 ± 0.1 165.2 ± 10 a

p value 0.0026 0.1889 <0.0001

Within column, means ± standard error indicated by different letters are significantly different,

Tukey Test p<0.05. CTRL= non-amended, covered control without sclerotia.

148

Table 4-3. Effect of carbon soil treatment on sclerotia of Sclerotium rolfsii in pot assay, C:N ratio growth chamber study, 2013

Carbon treatment

Sclerotial

germination

Trichoderma

parasitism of

sclerotia

Fusarium

parasitism of

sclerotia

Sclerotial

decomposition

Parasitism by

unidentified fungi

Parasitism by

fungi other than

Trichoderma

Percentage

Trial 1

CTRL 21.5 ± 5 a 81 ± 4.3 b 2.5 ± 1.4 2.5 ± 1.9 1.9 ± 1 b 4.4 ± 1.6

Dry molasses 11.2 ± 3.2 b 91.8 ± 2.6 a 0.7 ± 0.5 4.1 ± 1.4 4.5 ± 1.4 a 5.2 ± 1.4

Wheat bran 2.5 ± 1.1 c 96.1 ± 1.7 a 3.6 ± 2.1 2.2 ± 1.2 1.6 ± 0.8 b 5.2 ± 2.4

p value <0.0001 0.002 0.44 0.38 0.144 0.41

Trial 2

CTRL 38.5 ± 9 a 54.2 ± 9.2b 8.8 ± 2.8 5 ± 2.2 3.2 ± 1.5 12 ± 3.2

Dry molasses 3.8 ± 1.5 b 77.4 ± 5.2 a 11 ± 3.1 5.3 ± 2.3 13.3 ± 3.7 24.2 ± 5.2

Wheat bran 3.4 ± 2.6 b 84.8 ± 4.5 a 12.6 ± 3.7 4.1 ± 1.8 5.9 ± 1.8 18.5 ± 4.4

p value <0.0001 0.004 0.9 0.6 0.1 0.49

Within column, means ± standard error indicated by different letters are significantly different, Tukey Test p<0.05. CTRL= non-

amended, covered control without sclerotia.

149

Table 4-4. Growth characteristics of 8-week-old tomato plant transplanted at 5 weeks after

termination of ASD treatment sclerotia inoculated pots, carbon rate growth chamber study

Carbon ratea Mean fruit number Mean fruit Weight

per pot g pot-1

Trial 1 Trial 2 Trial 1 Trial 2

C-2 7.3 ±0.9 b 2.3 ± 1.1 164.9 ± 19.7 15.6 ± 10.5

C-4 17.7 ± 6.6 a 3.3 ± 1.8 159.1 ± 14.1 21.8 ± 11.9

C-6 6 ± 0.4 b 5 ± 1.8 138.5 ± 17 22.1 ± 7

C-8 5.5 ± 0.9 b 5.8 ± 2.1 136 ± 27.2 26.9 ± 10.3

CTRL 4.5 ± 1.9 b 1 ± 0.4 82.9 ± 9.3 10.1 ± 4.2

CTRL_S 8.3 ± 2.4 b 1 ± 0.7 152.2 ± 9.5 5.5 ± 3.2

C-0 6 ± 1.1 b 1 ± 1 130.7 ± 39.6 6.1 ± 6.1


Tukey Test p<0.05.

aCTRL=non-amended, uncovered control without sclerotia, CTRL_S=non-amended covered

control with sclerotia=C-2=Carbon rate 2 mg C g-1

soil, C-4=Carbon rate 4 mg C g-1

soil, C-

6=Carbon rate 6 mg C g-1


soil and C-0=Carbon rate 0 mg C g-1

soil

150

Table 4-5. Effect of C: N ratio soil treatment on soil gravimetric moisture content (g g-1

) in field

conditions

aTreatments are ASD10=C:N ratio 10:1, ASD20=C:N ratio 20:1, ASD30=C:N ratio 30:1,

ASD40=C:N ratio 40:1, LCASD30=C:N ratio 30:1, C rate 2 mg C g-1 soil (LC ± ‘low carbon’),

Fum= Fumigated control, (LC ± ‘low carbon’) and UTC= Untreated, non-amended control.


Tukey Test p<0.05.

C:Na

Field assay 1

Year 2013

Field assay 2

Year 2014

Pre ASD Post ASD Pre ASD Post ASD

ASD10 2.5 ± 0.16 2.9 ± 0.05 ab 2.7 ± 0.19 3.4 ± 0.24

ASD20 2.6 ± 0.09 3.1 ± 0.1 a 2.9 ± 0.11 2.9 ± 0.29

ASD30 2.7 ± 0.07 2.8 ± 0.15 b 3.1 ± 0.26 3.9 ± 0.55

ASD40 2.5 ± 0.05 2.5 ± 0.17 cd 3 ± 0.08 2.9 ± 0.35

Fum 2.5 ± 0.13 1.5 ± 0.53 d 2.7 ± 0.05 3.3 ± 0.33

LCASD30 2.7 ± 0.1 2.9 ± 0.08 ab 2.8 ± 0.11 3.1 ± 0.31

UTC 2.5 ± 0.05 2.8 ± 0.03 bc 2.8 ± 0.1 3.2 ± 0.29

151

Figure 4-1.Effect of amendment C:N ratio on soil pH (A), cumulative anaerobic condition (B),

percentage sclerotial germination (C), and percentage decomposed sclerotia (D) during ASD

treatment, pot study.

Bars indicated by different letters are significantly different at p<0.05 according to Tukey’s test.

Capital letters are used to compare the respective means of pot assay 1 and small letters are used

to compare respective means of pot assay 2. Error bars indicate standard error with four

replicates. CTRL=non-amended control, 10=C:N ratio 10:1, 20=C:N ratio 20:1, 30=C:N ratio

30:1 and 40=C:N ratio 40:1.

152

Figure 4-2. Effect of amendment C:N ratio on percentage sclerotial parasitism by Trichoderma

(A), Fusarium (B), other fungi (C), and Fusarium and other fungi (D) after ASD treatment, pot

study.


Capital letters are used to compare the respective means of the pot assay 1 and small letters are

used to compare respective means of the pot assay 2. Error bars indicate standard error with four

replicates. CTRL=non-amended control, 10=C:N ratio 10:1, 20=C:N ratio 20:1, 30=C:N ratio

30:1 and 40=C:N ratio 40:1

153

Figure 4-3. Effect of carbon amendments at depths 5-cm (top) and 15-cm (bottom) on percentage

sclerotial germination (A), percentage sclerotial parasitism by Trichoderma (B), Fusarium (C),

and other fungi (D) after ASD treatment, pot study.




replicates. CTRL=non-amended control

154

Figure 4-4. Effect of amendment C rates on soil pH (A), cumulative anaerobic condition (B),

percentage sclerotial germination (C), and percentage Trichoderma parasitism (D) during ASD

treatment, pot study.


Capital letters are used to compare the respective means of the trial 1 and small letters are used to

compare respective means of the trial 2. Error bars indicate standard error with four replicates.

CTRL=non-amended, uncovered control, C-2=Carbon rate 2 mg C g-1

soil, C-4=Carbon rate 4

mg C g-1

soil C-6=Carbon rate 6 mg C g-1


soil and C-0=Carbon

rate 0 mg C g-1

soil

155

Figure 4-5. Effect of amendment C rates on percentage sclerotial parasitism in pot trial 1 (A) and

trial 2 (B).


Capital letters are used to compare the respective means of the trial 1 and small letters are used to

compare respective means of the trial 2. Error bars indicate standard error with four replicates.

CTRL=non-amended uncovered control, C-2=Carbon rate 2 mg C g-1

soil, C-4=Carbon rate 4

mg C g-1



soil and C-

0=Carbon rate 0 mg C g-1

soil

156

Figure 4-6. Effect of dry molasses amendment at different C:N ratios on percentage sclerotial

germination (A), percentage sclerotial parasitism by Trichoderma (B) in the year 2013 (field

assay 1) and 2014 (field assay 2); percentage sclerotial parasitism other than Trichoderma in year

2013 (C) and 2014 (D) after ASD treatment.




replicates. ASD10=C:N ratio 10:1, ASD20=C:N ratio 20:1, ASD30=C:N ratio 30:1,


and UTC=Untreated, non-amended control.

157

Chapter 5

Assessment of beneficial microorganisms: Trichoderma,

actinomycetes, Bacillus and root colonizers in anaerobic soil

disinfestation.

158

A version of this chapter is a manuscript in preparation for Phytopathology by Utsala Shrestha,

Mary Dee, Bonnie H. Ownley and David M. Butler.


analysis, results interpretation and writing. Mary Dee helped in examination of sclerotial

germination, parasitism and soil serial dilutions of beneficial organisms.

159

Abstract

Studies on anaerobic soil disinfestation (ASD), a non-chemical alternative to soil fumigants for

controlling many soilborne diseases, have shown that it enhances populations of beneficial

microorganisms against plant pathogens, including increased presence of the biocontrol agent

Trichoderma as sclerotial parasites of Sclerotium rolfsii. However, studies on ASD effectiveness

paired with beneficial mycoparasites and commercial biofungicide applications are lacking. This

study compared the effect of ASD and incorporation of antagonists separately or in combination,

at the initiation of ASD treatment, against the sclerotial germination and parasitism. The effect of

ASD amendment on soil populations of endophytic isolates of Trichoderma, actinomycetes, and

Bacillus spp. were also assessed. The anaerobic condition was also determined during ASD

treatment in growth chamber studies. The root nodules of cowpea and plant biomass (cowpea

and tomato) after ASD treatment were also recorded in greenhouse study. Arbuscular

mycorrhizae were quantified from pepper plant in field study. In contrast to the negative effect of

ASD on sclerotial population, we observed positive or no effect on the population of beneficial

microorganisms. Further, ASD enhanced the mycoparasitic and bacterial colonization of

sclerotia; however, ASD followed by addition of antagonists did not increase sclerotial mortality

or parasitism of sclerotia.

Keywords: Actinomycetes, arbuscular mycorrhizae, anaerobic soil disinfestation, Bacillus,

cowpea, Mycostop®

, nodules, parasitism, RootShield®

, sclerotia, Trichoderma, tomato

160

1. Introduction

Worldwide awareness in sustainable and chemical-free farming and food concepts has

encouraged farmers to pursue the best non-chemical techniques to control pests. The ASD

approach to soil disinfestation reduces chemical pollutants, reduces human health risks, and

enhances safety of farmers and residential areas. Optimizations of ASD with various organic

amendments at different rates have been examined in varied temperature regimes against

different soilborne pathogens (Fusarium, Ralstonia, Rhizoctonia, Verticillium, and

Phytophthora). ASD also induces changes in soil physical characteristics especially, soil pH, soil

moisture and soil nutrients, due to addition of organic matter (Bonanomi et al., 2010). It is also

reported to cause a significant shift in the microbial community composition (Mazzola et al.,

2012), especially the composition of the bacterial community or soil aerobes (Messiha et al.,

2007; van Agtmaal et al., 2015), and does not re-establish the original community structure

during aerobic incubation, following ASD treatment. Bacterial activity in the early stage of soil

treatment by ASD is considered more important for ASD effectiveness than in the later stages,

emphasizing the fact that indigenous microorganisms play important roles in the biological

control capacity of ASD. Anaerobic microorganisms, including Bacillus and Clostridium spp.,

are well-known antibiotic and toxin producers and bacterial community studies, particularly

Clostridia species, have suggested that their effectiveness to suppress soilborne diseases

(Momma et al., 2013; Mowlick et al., 2013a; Huang et al., 2016), is due to production of volatile

fatty acids (Mowlick et al., 2013b). Hong et al. (2013) revealed that ASD treatment increases

beneficial populations of the bacterial genera Bacillus and Paenibacillus, that may act as

biocontrol agents in ASD.

Soil microbial community also comprises fungal mycoparasites and endophytic beneficial

microbes; however, fungal activities and their interaction with the ASD treatment or with

beneficial populations have not been investigated in detail. Studies on Trichoderma spp. as

biocontrol fungi against soilborne diseases (Chet et al., 1979; Kredics et al., 2003) have been

extensively studied (Lewis and Papavizas, 1983; Lewis and Papavizas, 1991), and Trichoderma

spp. are commercially used to control soilborne pathogens such as Fusarium, Pythium,

Rhizoctonia, Sclerotinia, Alternaria, and more (see Verma et al. (2007), Table 1). Various

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Trichoderma spp. have been studied as sclerotial parasites (Papavizas and Lewis, 1989; John et

al., 2015; Pacheco et al., 2016). Trichoderma is also present as a sclerotial parasite after ASD

treatment (McCarty, 2012; Shrestha et al., 2013; Rosskopf et al., 2015). Organic amendments

play important role in the proliferation of Trichoderma (Yossen et al., 2008; Bonanomi et al.,

2010) and ASD treated soil might also support its growth. However, successful soil treatment by

ASD is a function of change in soil pH, metal ions, and facultative and anaerobic bacteria. These

parameters are also known to affect the biocontrol efficacy of Trichoderma (Duffy et al., 1997;

Kredics et al., 2003), and better understanding of how these parameters affect biocontrol with

Trichoderma in ASD would be helpful, while optimizing the ASD amendment sources.

Trichoderma harzianum applied to tomato transplants in combination with Bacillus subtilis

resulted in greater control of soilborne diseases and yield of tomato, than when used singly

(Morsy et al., 2009). However, Trichoderma is highly competitive with other soil microbes

(Kaur et al., 2005), and its effect on other potential biocontrol organisms during ASD treatment

is unknown. In previous ASD studies, isolates of bacteria Bacillus, actinomycetes, Trichoderma,

zygomycetes and nonpathogenic endophytic Fusarium were recovered from field soil following

ASD treatment (Shrestha et al., 2013) and found Bacillus and actinomycetes (Streptomyces spp.)

as new biocontrol for sclerotial parasites (Adhilakshmi et al., 2014; Gholami et al., 2014). Our

hypothesis for this study was that integration of these potential biocontrol agents along with

carbon amendments into the ASD system will improve effectiveness of biocontrol against

soilborne plant pathogens.

Many Trichoderma spp. have been identified and used as active ingredient of commercial bio-

pesticides (Woo et al., 2014). In USA, several Trichoderma based products have been registered

for crop protection such as: T. hamatum, Floragard (Sellew Associates, LLC); T. harzianum DB

103, T-Gro (Dagutat Biolab); T. harzianum Rifai Strain T-22, RootShield® WP biological

fungicide; T. virens strain G-41, BW240 G, BW240 (Bioworks inc); T. polysporum Rifai ATTC

20475 and T. viride sensu Bisby , T. viride ATCC 20476 (Binab Bioinnovation eftr ab ). There

are several other Trichoderma species based product that is under registration process. Among

various spp., T. asperellum has been identified as a potent biopesticides against various

pathogens and was recently commercialized as active ingredients under the commercial name

Ecohope, Ecohope-Dry (Japan), Quality WG, Trichodermax EC (Brazil) and Trichotech

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(Kenya). Recognizing the commercial importance of biofungicidal properties of T. asperellum,

and we selected local isolate of T. asperellum recovered from S. rolfsii sclerotia as one of our

antagonists in this study. Two commercially available biofungicides (Table 1) with active spores

of Trichoderma harzianum (RootShield®

) and Streptomyces riseoviridis (Mycostop®

) were

selected to incorporate in soil with ASD treatment to see how these antagonists impact the

efficacy of ASD. We determined the impact of these biocontrols by analyzing the germination

and parasitism of sclerotia of S. rolfsii in ASD with C rates of 4 mg C g-1

at C:N ratio 30:1. In

addition, we also evaluated the impact of ASD amendment on soil populations of Trichoderma,

actinomycetes, and Bacillus.

Rhizobia are nitrogen-fixing beneficial microbes that induce nodule formation where

atmospheric nitrogen is fixed (Shantharam and Mattoo, 1997). To gain insights into the effect of

ASD on indigenous rhizobium populations, we quantified the number and mass of cowpea root

nodules because nodule mass is directly related to bacteroid numbers (Wadisirisuk and Weaver,

1985). The symbiotic association between arbuscular mycorrhizal (AM) fungi and pepper root is

reported to increase plant growth as AM fungi helps in uptake of phosphorus nutrient in

exchange for photosynthates (Davies et al., 1992; Martin and Stutz, 2004). Very little is known

about the impact of ASD on AM fungi. To determine the ASD effect on this beneficial organism,

we quantified total root colonization by AM fungi on pepper after ASD treatment.

2. Material and Methods

2.1 Sclerotia inoculum preparation

An isolate of S. rolfsii from tomatoes was grown in petri dishes (100 mm x 15 mm) with full-

strength potato dextrose agar (PDA; BD BBL™, Fisher Scientific) for 7 days, and plugs were

transferred to partitioned Quad PDA plate. Cultures were then allowed to grow to obtain mature

sclerotia as described in Chapter 4. Mature sclerotia were harvested from PDA plates after 1 to 2

weeks. Sclerotia were placed in 5-cm × 5-cm bags made from Delnet® aperture film. The film

allows water, and air to pass through. Bags containing 10 sclerotia were prepared for different

depth before ASD treatment. Bag containing 100 loose sclerotia were prepared for burial at 2-cm

depth.

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2.2 Isolation and spore suspension of Trichoderma asperellum

The culture of T. asperellum was isolated from parasitized sclerotia in soil previously treated

with ASD. Trichoderma asperellum was identified by extracting genomic DNA from colonies

grown on potato dextrose broth for at least a week using the Qiagen plant extraction kit.

Elongation factor 1 (EF1) and EF2 regions were amplified by PCR and the amplicon was

sequenced. The sequences obtained were used to blast in the NCBI database. The EF1and EF2

sequences were 99% identical to those of GenBank accessions of T. asperellum. Spore

suspensions from T. asperellum isolates were prepared in double deionized water by harvesting

green spores from a 2-week-old culture. Number of spores was quantified using a

haematocytometer and then diluted to obtained 1.3 x 105spores ml

-1.

2.3 ASD treatments and/or biocontrol treatments

Dry molasses (C:N~29.7, Westway, New Orleans, LA) that was mixed with corn starch (C:N~0,

Tate & Lyle ingredients Americas, INC. Decatur, IL) was adjusted to a C:N ratio 30:1 of ASD

treatments. Two commercial biocontrol agents RootShield®

(Bioworks Inc. Geneva, New York,

USA) and Mycostop®

(Verdera, Espoo, Finland) and T. asperellum isolated from parasitized

sclerotia were selected to evaluate impact of ASD on biocontrol populations (Table 1). ASD

treatments included combination of ASD with i) RootShield®

, ii) Mycostop®

, iii) RootShield®

and Mycostop®

and iv) T. asperellum and non-ASD treatments included i) RootShield®

, ii)

Mycostop®

, iii) RootShield®

and Mycostop®

and iv) T. asperellum. Amended ASD control and

two non-amended controls without carbon supplement, with and without plastic covering, were

included.

2.4 Pot setup

Growth chamber study conditions were maintained similar to previous studies (25°C for 14 h,

15°C for 10 h with 50% relative humidity). ASD was was carried out for 3 weeks with soil

(Fine-loamy, siliceous, semiactive, mesic Typic Hapludult ) collected from the ‘Ap’ horizon at

the Plateau Research and Education Center, Crossville, TN, where wheat was planted. Soil to fill

20-cm square polyethylene pots (~10.2-cm width x 24.1cm height, 1.5 L) was sieved (<10 mm)

to remove organic debris and mixed with white fine sand at 1:1 proportion (pH 6, trial1 and pH

http://blast.ncbi.nlm.nih.gov/Blast.cgi#alnHdr_169666124

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6.7, trial 2). Soil mixture was mixed with dry molasses and corn starch and placed in pots. Three

bags with ten sclerotia of S. rolfsii each were buried at 5-, 10- and 15-cm depths in each pot.

Bags with 100 sclerotia were buried at 2-cm depth. Oxidation-reduction electrodes (ORP) and

temperature-moisture sensors were inserted at 10- to 15-cm depths to measure cumulative redox

potential and temperature of soil. Three replicates of each treatment received ORP probes and

two replicates received temperature probes. Pots for each biocontrol treatments were carefully

drenched with ~500 ml spore suspension prepared in sterile deionized water to attain complete

saturation. Other pots were saturated with deionized water. All pots were covered with black

polyethylene (0.03 mm) with a heavy-duty rubber band, except a non-amended treatment for 3

weeks. The design was completely randomized with four replicates that started on the 5 to 25 of

March 2015 and was repeated on 24 June to 15 July 2015.

When ASD was completed, plastic, ORP, and temperature probes were removed. Bags with

string were carefully removed and soil samples adhered to each bags along with soil within 15-

cm depth were collected in sampling bag. A soil samples was collected for estimation of

biocontrol agents populations. Subsamples were oven-dried (105°C for 48 h) to determine

gravimetric moisture content and the remaining sample was air-dried and sieved (<2 mm) for soil

pH. Soil pH and Cumulative soil anaerobic condition was calculated as described previously

(Butler et al., 2012b; McCarty et al., 2014).

2.5 Pathogenicity testing

The top bag of each pot was opened and sclerotia were mixed with the top 2-cm soil. Pots were

moved to the greenhouse bench and arrranged in randomized block design with four replications.

The mean temperature of the greenhouse and the mean relative humidity of the greenhouse was

25°C (ranging 18 to 38°C ) and 55% during trial 1 and 30°C (39 to 23°C) and 90% during trial 2,

respectively. Three-week-old tomato plants (cv. Florida Lanai) and two pre-sprouted cowpea

seeds (cv. California black-eyed pea) that were later thinned to one were planted in each pot.

Sclerotial survival and disease pressure were evaluated weekly on plants and the disease severity

index was measured for tomato and cowpea plants as described in Guzmán-Valle et al. (2014)

and Errakhi et al. (2007), respectively. The nominal scale used to quantify disease severity was 1

= healthy plants, 2 = plants without symptoms but with sclerotia in the soil, 3 = yellowish

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colored plants with surrounding mycelium and sclerotia, 4 = plants with symptoms of wilt and

rot with mycelium and sclerotia. Each pot was drip irrigated 1 min for the first week and then

increased to 2 min every morning to maintain proper moisture content. After 8 weeks, shoots and

roots of tomato and cowpea plants from each pot were oven-dried at 65°C for dry biomass.

Before drying cowpea roots, root nodules were cleaned with tap water, counted and dried at

room temperature before taking the weight.

2.6 Assessment of sclerotial germination and parasitism

To access germination and parasitism of sclerotia, bags of sclerotia were retrieved from bags at

5-, 10- and 15-cm depths, and sclerotia were plated onto 24 well plates containing i. PDA

(Difco™ Potato Dextrose Agar) amended with 6.9 mg l-1

fenpropathrin (Danitol 2.4 EC, Valent

Chemical, Walnut Creek, CA) and 10 mg l-1

rifampicin (Sigma-Aldrich, St. Louis, MO), ii. AIA

(Difco™ Actinomycete Isolation Agar, 22 g l-1

) mixed with 5g Difco™ Glycerol, and iii. TSM

(Trichoderma selective medium) Trichoderma modified PDA adapted from Gil et al. (2009)

containing 39 g l-1

PDA amended with 0.02 g l-1

, rose bengal, 0.3 g l-1

chloramphenicol, 0.02 g l-1

streptomycin sulfate maintained at pH 6 (Figure 5-1). We used different media for different

depths as our objective was to only access the sclerotial germination and parasitism on a

respective medium and not intended to make any comparison among depth of inoculation. Plates

were incubated at room temperature for at least 3 weeks and observation were made for mycelial

growth, germination of sclerotia, parasitism of sclerotia by Trichoderma, zygomycetes,

Fusarium spp. and actinomycetes or other bacteria.

2.7 Quantification of Trichoderma, actinomycetes, and Bacillus from soil

Media plates for quantification of Trichoderma and actinomycetes were prepared using TSM and

AIA. Media plates for Bacillus, facultative anaerobic spore formers (Bacillus isolation medium =

BIA), were prepared by adding 15 g potato dextrose agar, 5 g glucose (Dextrose), 5 g peptone, 3

g beef extract and 1 g yeast extract. One gram of composited soil samples from each pot were

suspended in 9 ml of sterile double-deionized water and serial dilutions from 10-2

to 10-4

were

prepared for Trichoderma and actinomycetes, and dilutions of 10-3

to 10-6

were prepared for

Bacillus. An aliquot of 0.1 ml of each dilution was spread on plates containing selective media

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until liquid was absorbed into the TSM and AIA media. Before plating Bacillus in BIA, serial

dilution tubes were heated (80 to 85°C) in a water bath for 30 min to activate heat-resistant

spores and kill non-heat tolerant vegetative cells in the soil sample, and then spread onto agar

plates. Inoculated plates for three biocontrols were duplicated and incubated at room temperature

for 18 to 24 hr for Bacillus, 1 to 2 weeks for Trichoderma and 4 to 6 weeks for actinomycetes.

Emerging colony forming units (CFU) of each biocontrols from each plates were counted and

expressed as CFU per gram of soil, and re-isolated in selective medium for identification (Figure

5-2). Trichoderma isolates were preserved in slant amended PDA tubes. Fifty percent (v/v)

glycerol stock solution was prepared for preservation of actinomycetes in actinomycetes

vegitone broth (AVB) and Bacillus in Nutrient broth yeast extract (NBY), and was stored at -

20°C for future study.

2.8 Molecular identification

To identify Trichoderma, the fungus with green appearance on PDA was observed under the

microscope. Further identification of Trichoderma spp. was done by extracting genomic DNA

from colonies of the isolate grown on PD broth using the QiagenDNeasy DNA extraction kit.

ITS regions 1 and 2 of Trichoderma isolates (White et al., 1990) amplified by PCR using the

appropriate primers pairs (5'-TCCGTAGGTGAACCTGCGG-3' / ITS2: 5'-

GCTGCGTTCTTCATCGATGC-3'). PCR was carried out in a 50-μl reaction mixture containing

50 ng genomic DNA, 5 μl each of 0.5 μM forward and reverse primers, 1 μL dimethylsulfoxide

and 25 μL of 5 PRIME HotMasterMix (VWR International). The PCR conditions was an initial

denaturation of 94°C for 2 min followed by 42 cycles of 1 min denaturation at 94

°C, 1 min of

annealing at 420C and 2 min extension at 72

°C, and final extension of 3 min at 72

°C. The PCR

products was purified using ExoSAP-IT®

PCR Product Cleanup ExoSAP-IT (Affymetrix, Santa

Clara, CA), and sequencing was done by the Molecular Biology Resource Facility, UTK,

Knoxville, TN. The resultant sequences were used to blast in the NCBI GenBank database and

were edited with Sequncher (v 5.1) to configure for highest accuracy.

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2.9 ASD effect on AM fungi

To quantify the root colonization of pepper root, three root systems at the end of harvesting

selected randomly from the field experiment at the UT Plateau Research and Education Center in

Crossville (see experiment layout in chapter 3). Roots were cleaned with tap water and stored at

4°C until analyzed. The modified staining procedure of Grace and Stribley (1991) was followed

to stain the root samples. Briefly, cleaned root samples were cut into 1-cm pieces to fit in a

histology tissue cassette and boiled at 70-80°C in KOH (10%) solution for 10-15 min. The roots

were cooled at room temperature and then rinsed with tap water before acidifying with HCl (2%)

for 1.5 hr. Roots were stained in trypan blue (0.05%) for 1 hour. Cassettes with root were rinsed

2-3 times with distilled water and then immersed in lactoglycerol to destain. The root segments

were then observed under the microspce to visualize AM fungal hyphae, vesicles, and or

arbuscules.

2.10 Data analysis

Data was subjected to analysis of variance using mixed model in SAS (Glimmix procedure, SAS

Institute, Cary, NC) to find the relationship between applied beneficial organisms and anaerobic

conditions in ASD, and to determine the impact on sclerotial germination and crop performance.

Our design was randomized compete block design with trial as a random factor and treatments as

a fixed factor. DNA sequences were edited using Sequencher 5.0 (GeneCodes). These sequences

were compared with sequences in the NCBI GenBank nucleotide database to identify the

bacterial or fungal species. Sequences were aligned using ClustalX (Larkin et al., 2007).

Phylogenetic analysis was done using the program MEGA 5.2 (Tamura et al., 2012).

3. Results

In our previous field study, ASD treatment with 4 mg C g-1

soil used at 30:1 C:N ratio was the

most effective in increasing sclerotial mortality, although, we observed no significant difference

among C:N treatments in both pot and field studies. In this study, we tested wheather the

effectiveness of ASD against sclerotia could be improved by incorporating commercial bio-

fungicide or endemic Trichoderma populations before ASD treatment. We also performed serial

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soil dilutions on ASD treated soil to observe the effect of ASD on populations of beneficial

microbes. We observed a significant weak negative relationship between sclerotial germination

with cumulative anaerobic condition and sclerotial parasitism by Trichoderma, zygomycetes and

bacteria (-0.3 to -0.5, p<0.001). Trichoderma parasitism also had a positive weak relationship

with soil pH and cumulative anaerobic condition. Parasitism (%) by ”other fungi” and bacterial

parasitism showed moderate positive relationships and both showed a negative relationship with

soil pH and soil moisture (Table 5-2).

Cumulative redox potential (Ceh) was measured as an indicator of cumulative anaerobic

conditions. We observed that the ASD treatments had Ceh readings of 128,858 to 141,019 mV hr

which were significantly higher than non-ASD treatments (1,033 to 29,464 mV hr), providing

evidence for the generation of the anaerobic condition in ASD treatments (Figure 5-3A). Soil pH

ranged from 6.12 to 6.61 units across treatments and theses were significantly higher soil pH

values for T. asperellum.Soil pH was lowest for untreated controls and non-ASD treatments

except for Mycostop®

+RootShield® (Figure 5-3B). The post gravimetric soil moisture content

was significantly higher in the covered control, ASD + Mycostop®

, and Mycostop®

treatments

(0.18 g g-1

) than other treatments (~0.16g g-1

) and lowest for the uncovered, unirrigated control

(0.03 g g-1

).

3.1 Germination of sclerotia

Previously we observed that ASD is effective in increasing sclerotial mortality. In order to test if

the incorporation of antagonists such as Mycostop®

or RootShield®

or the endophytic isolates (T.

asperellum) could further improve effectiveness of ASD, sclerotia were inoculated in soil at

different depths. After ASD completion, sclerotial germination was tested. We observed that

germination of sclerotia retrieved from ASD treatments from 5-, 10- and 15-cm depth were, in

general, significantly lower than non-ASD treatments (Figure 5-4). At 15-cm, sclerotial

germination from non-ASD RootShield®

treatment was significantly lower and similar to ASD

treatments (0 to 7.5%). Trichoderma. asperellum with ASD had a lower percentage of

germinated sclerotia (14%) than non-ASD T. asperellum (33%), but it was not significant.

Mycostop®

alone, in non-ASD, failed to suppress sclerotia and was similar to the covered control

(63-67%). There were no significant differences in the suppression of sclerotial germination

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among the ASD treatments (Figure 5-4C). Similarly, at the 10-cm depth, all ASD treatments

alone or with inoculated antagonists, except Mycostop®

, significantly contributed to sclerotial

mortality (0-5%). The non-ASD treatments with biofungicides were not effective in sclerotial

suppression (18-30%) and had the highest germination recorded for T. asperellum (38%, Figure

5-4B). At greater depth (15-cm) under non-ASD conditions, addition of organic amendments

plays an important role in sclerotial suppression.Addition of Mycostop®

biofungicides and

endophytic Trichoderma under anaerobic condition were not effective, and may have been

affected adversely by high gravimetric soil moisture (Figure 5-3C) and gravitational pressure.

Interestingly, at 5-cm depth sclerotial germination in the ASD only treatment (18%) was lower

than ASD treatments into which other anatagonists were also added. At 5-cm, mortality of

sclerotia was lower than greater depth (10-15-cm) and use of only anatagonists did not reduce

sclerotial germination compared to the plastic covered or non-covered control (57-75%). At 5-

cm, we observed that sclerotial germination was highest for Mycostop®

+ RootShield®

(88%)

followed by two controls and non-ASD T. asperellum. Our results showed that the use of

commercial bio-fungicides such as Mycostop®

or RootShield®

, or endophytic isolate - T.

asperullum did not reduce sclerotial germination under covered condition at any depths. Addition

of these antagonists into ASD also did not promote further suppression of sclerotial germination,

compared to ASD only treatment.

3.2 Sclerotial parasitism

Sclerotial bags at 5-, 10- and 15-cm depths were retrieved and cultured in PDA, AIA and TSM,

to test sclerotial parasitism by fungi and bacteria parasites. Although our objective was not to test

the sclerotial germination or parasitism differences across different depths or media, we observed

that Trichoderma, zygomycetes, bacteria and other fungal parasites of sclerotia occured across

soil of depths of 5-, 10- and 15-cm. However, sclerotial parasitism by actinomycetes was only

observed in sclerotia retrieved from 10-cm and plated on AIA plates.

Sclerotia retrieved from 5-cm depth plated on PDA showed relatively higher percentage

Trichoderma parasitism of sclerotia in ASD treatments compared to the control. Incorporation of

antagonists with ASD did not increase sclerotial parasitism by Trichoderma. Sclerotial

parasitism by Trichoderma in these treatments with antagonist was equivalent to their

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corresponding antagonists in non-ASD treatments except treatment that used both Mycostop®

and RootShield®

(Figure 5-5A). Our results showed that sclerotial parasitism by zygomycetes in

ASD treatments with antagonists were higher in non-ASD treatments with corresponding

antagonists. For sclerotia retrieved from 5-cm, parasitism by other fungi was highest for the

control, but we did not observe any significant difference among any treatments in sclerotial

parasitism by bacteria.

Sclerotia retrieved from 10-cm, plated on AIA (Figure 5-7) showed significantly highest

bacterial parasitism in ASD alone and ASD with antagonists, except Mycostop®

, when compared

with non-ASD with antagonists (72-83%). Zygomycetes parasitism of sclerotia was significantly

greater in all ASD treatment with the highest parasitism observed in ASD + Mycostop®

(98%)

compared with both non-ASD and control treatments. In contrast, Trichoderma colonization of

sclerotia was significantly higher in non-ASD treatment and the control (45-73%,). Figure 5-6

represents the actinomycete parasitism of sclerotia, which was significantly higher in Mycostop®

treatments (65-88%) even though we observed high percentage of sclerotial germination. This

also suggests that actinomycetes from addition of Mycostop®

competed with other

mycoparasites, especially Trichoderma, bacteria and other fungi for sclerotial parasitism in ASD

treatments. This study does not provide any evidence of significant parasitism of sclerotia by

endemic actinomycetes in ASD. These results indicate the significance of using isolation media

such as AIA for actinomycetes and bacterial parasitism of sclerotia.

Surprisingly, sclerotial parasitism on TSM, i.e. sclerotia retrieved from the 15-cm depth showed

significantly higher Trichoderma parasitism of sclerotia in non-ASD and control (32-72%) than

ASD treatment with or without antagonists. The zygomycetes parasitism of sclerotia was

significantly higher in ASD alone (40%) and addition of the antagonists increased sclerotial

parasitism by zygomycetes for ASD with Mycostop®

+Rootshield®

. Intriguingly, we observed

significant sclerotial parasitism by other fungi (for e.g., Myrothecium spp., Penicillium spp.) at

15-cm for ASD.We not only observed highest mortality of sclerotia at 15-cm depth but also

noticed limited Trichoderma colonization of sclerotia in all ASD treatments revealing an

unfavorable environment for Trichoderma at greater depth (Figure 5-8).

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3.4 Effects on beneficial organisms

Our previous studies showed that ASD had negative effects on soilborne pathogens and altered

microbial composition. Addition of relatively high amendment rate under covered and irrigated

condition induces the anaerobic environment and alters soil properties as well (Inglett et al.,

2005; Butler et al., 2014) with the production of organic acids (Momma et al., 2006) and other

volatiles (Hewavitharana et al., 2014). Therefore, we were interested to determine the impact of

ASD on soil beneficial microorganisms. To test the effect of ASD on beneficial microorganisms,

such as actinomycetes, Bacillus and Trichoderma, we counted the number of colony forming

units with standard dilution plating of soil samples on selective media. Our analysis showed that

ASD had no effect or a positive effect on the beneficial microorganism populations. We

observed that use of different antagonists reduced the populations of actinomycetes during ASD

treatment (Figure 5-9). We observed enhanced populations of Bacillus in ASD treatments with

highest CFU recorded for ASD with T. asperellum and the combination of Mycostop®

and

RootShield® (5.6 log CFU+1 g

-1 of soil, Figure 5-10). Trichoderma spp. populations were similar

across the ASD treatments and was highest in ASD when compared to ASD with antagonist,

except for T. asperellum with and without ASD (Figure 5-11). Different endophytic fungi

identified from soil are presented in Figure 5-16.

3.5 Effect on nodule and colonization of AM

We observed that cowpea root nodule number and mass did not change in ASD treatment

compared to the controls suggesting that ASD has no negative effect on the population of

rhizobium. We also observed that incorporation of T. asperellum enhanced cowpea nodule mass

compared to the controls under ASD conditions indicating an enhanced symbiotic relationship

among cowpea root, T. asperellum, and indigenous nodule forming bacteria (Figure 5-13). Root

colonization by AM was significantly higher in all ASD treatments with 4 mg C g-1

of soil in the

field study (Figure 5-14), when compared to fumigated and non-amended plots.

3.6 Effect on root and shoot biomass

There was no significant difference in the total root biomass of cowpea and tomato among all

non-ASD treatments and control treatments. Only ASD with T. asperellum showed higher root

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biomass when compared to ASD + Rootshield®

, non-ASD treatments with antagonists and

control treatments. Assessment of dry shoot biomass showed higher biomass only in ASD with

T. asperellum (17 g/pot) compared to ASD, ASD + Mycostop®

+ RootShield®

and non-ASD and

control treatments.

4. Discussion

Anaerobic soil disinfestation has been widely studied to control soilborne pathogens and it has

been demonstrated that the ASD is efficient for control of some nematodes and weed propagules

and ultimately contributes to yield increment (increases when combined with solarization) when

compared to non-amended controls (Butler et al., 2012a). Nevertheless, the pepper yield data

from Chapter 3 had significantly higher marketable yield than both fumigated and control

treatments. Besides pathogen control and yield, many studies have focused on alternation in

microbial community composition during ASD treatment (Mowlick et al., 2012; Streminska et

al., 2014; van Agtmaal et al., 2015; Huang et al., 2016).These studies used ethanol, wheat bran,

crucifers, commercial protein-rich vegetal by-product (Herbie®

) as carbon amendments for ASD.

The microbial study of ASD is of interest as temporary anaerobic condition generated during the

process stressed the microbial population and resulted in the microbial shift which risks the

pathogen suppressive ability of soil (van Agtmaal et al., 2015). These studies mainly focused on

bacterial composition and results showed increased populations of Bactereroidetes, Bacillus, and

Clostridia spp. Only a few studies have focused on endemic soil populations of beneficial fungi.

Trichoderma spp. and endemic Fusarium spp. are predominant after organic acid addition in

ASD (Rosskopf et al., 2014); however, results were not consisted among during trials. Since our

amendment in this study was dry molasses, it is noteworthy to see an effect of ASD on the

endemic population of Trichoderma and other potential sclerotial parasites, such as

actinomycetes and Bacillus. Biological based pesticides coupled with other pathogen control

mechanisms have shown promising results in pathogen suppression (Cook and Baker, 1983). The

Trichoderma based biopesticides are available worldwide (Woo et al., 2006; Woo et al., 2014)

and are primarily used for seed and transplant treatments. Similarly, Mycostop®

is also marketed

as a seed dressing or soil treatment (White et al., 1990). For our study, we selected RootShield®

with fungal spores of T. harzianum Rifai Strain T-22, and Mycostop®

with actinomycetelspores

173

of Streptomyces riseoviridis and active spores of the sclerotial mycoparasite T. asperellum i.e.

isolated and grown on PDA.

Application of biofungicides and endophytic mycoparasites before ASD treatment, in

combination with irrigation supplement, successfully suppressed sclerotial germination when

compared with antagonists added to pots with non-ASD conditions (i.e. non-amended , irrigated

and covered) and control treatments. However, the lowest sclerotial germination suppression was

observed at 5-cm depth as compared to 10-cm and 15-cm. Individual addition of Mycostop®

at

10-cm and T. asperellum at 15-cm in ASD did not improve the efficacy of ASD against

Sclerotium germination. At all depths, regardless of added antagonists, the sclerotial parasitism

by Trichoderma, zygomycetes and bacteria were apparent, but actinomycete parasitism was only

observed in AIA medium. After 3-weeks of incubation of soil under ASD treatment, populations

of beneficial soil organisms, Trichoderma and Bacillus increased and there were no negative

effects on actinomycetes observed. However, addition of antagonists in ASD treatment impacted

the filamentous spore-formers population and we hypothesize that actinomycetel spores might

retard their growth and failed to compete with other mycoparasites. In soil alone, actinomycetes

(especially Streptomyces spp.) are considered poor competitors when they do not colonize the

plant root (Lahdenperä et al., 1991; Lahdenpera, 2000). Actinomycetes populations under

anaerobic conditions increase their population when chitin is present; however, due to presence

of high organic matter, active chitin decomposers, such as Trichoderma, may compete with

actinomycetes under anaerobic condition (Manucharova et al., 2006). Chitinase and glucanase,

which is reported to control S. rolfsii (El-Katatny et al., 2001) from Trichoderma spp. inhibit S.

rolfsii by cell wall lysis (Elad et al., 1983; Sivan and Chet, 1989; Chet and Inbar, 1994; El-

Katatny et al., 2000). Bacillus, on the other hand, is a facultative anaerobe and previous studies

showed that Bacillus is present in soil treated with ASD (Mowlick et al., 2013a). Trichoderma

spp. colonization of sclerotia induced greater mortality of sclerotia in ASD alone or ASD in

combination with antagonists. Soil Trichoderma populations were also highest in ASD- treated

soil. Besides Trichoderma, we observed significant contribution of zygomycetes as

mycoparastism. Zygomycetes have been isolated from sclerotia of Sclerotinia sclerotiorum

(Ćosić et al., 2012) and produce glucanase and chitinase (Elad et al., 1985). Surprisingly,

Trichoderma and zygomycetes grow slowly in anaerobic conditions, however, these fungi are

174

active and in the presence of organic matter, they revive and recover in aerobic conditions

indicating a facultative nature. In addition, after isolation of these organisms from anaerobic

conditions their relative abundance increases (Kurakov et al., 2008).

Growers are concerned about the phytotoxic effect of ASD on root growth (McCarty et al., 2014;

van Agtmaal et al., 2015) and our study showed no negative impact of ASD on root growth.

Further, it does not influence nodule formation in cowpea and amendment applied during ASD

treatment may have enhanced root colonization of AM fungi in pepper plant (Gosling et al.,

2006), and may have contributed to yield increment (see Figure 3-7). Antagonists addition before

ASD did not enhanced the total root and shoot biomass, although the presence of Trichoderma

populations is supposed to enhance plant growth (Harman et al., 2004). Overall, we did not see

any significant impact on plant growth in controls, compared to non-ASD treatments, even

though we added 100 sclerotia in the top 2-cm of soil before ASD treatment. Soil in pots may

have enhanced beneficial organisms as these might be opportunistically feeding on sclerotia that

were feebled by the absence of a host infection zone during the three-week period.

5. Conclusion

We observed no effect of antagonists when incorporated during ASD treatment. Therefore, we

suggest studying the effect of the antagonist in ASD by adding antagonist after the completion of

ASD. Identification of Bacillus and actinomycete species is ongoing and quantification of the T.

harzianum population with qPCR might further elucidate the Trichoderma parasitism and actual

quantification of the Trichoderma population. Further research on the impact of ASD on

beneficial organisms in field condition could enhance the effects of ASD and carbon

amendments on plant disease.

6. Acknowledgements

Research supported in part by funding from the U.S. Department of Agriculture, Southern SARE

grant GS14-128, and National Institute of Food and Agriculture, U.S. Department of Agriculture

under award number 2012-51102-20293 (Methyl Bromide Transitions Program).

175

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181

Appendix

Table 5-1. List of biocontrol agents and ASD treatment

Biocontrol agents Active ingredients Concentration g-1

Application rate

Trichodema

asperellum

T. asperellum isolated from

parasitized sclerotia 1.3 x 10

5CFU ml

-1

1,000 spores

sclerotium-1

RootShield®

T. harzianumRifai strain

KRL-AG2 (1.15%) 3,000 CFU ml

-1 0.3 g l

-1

Mycostop®

Streptomyces Strain K61

(4%) 1,000 CFU ml

-1 0.01 g l

-1

RootShield®

+

Mycostop®

T. harzianumRifai strain

KRL-AG2 (1.15%) +

Streptomyces strain K61

1,500 + 500 CFU

ml-1

0.15 + 0.005 g l

-1

182

Table 5-2. Correlation among sclerotial germination, sclerotial parasitism and soil properties

during ASD treatment, pot study, 2015

Variables Trichoderma

parasitism

Zygomycetes

parasitism

Other fungal

parasitism

Bacterial

parasitism

Total

parasitism* Soil pH Ceh

Post soil

moisture

Sclerotial

Germination@

-0.46 -0.33 0.06 -0.32 -0.09 0.05 -0.47 0.01

<0.0001 0.002 0.610 0.003 0.388 0.628 <0.0001 0.911

Trichoderma

parasitism

-0.04 -0.26 -0.17 -0.15 0.25 0.23 0.20

0.678 0.014 0.123 0.171 0.022 0.046 0.062

Zygomycetes

parasitism

-0.09 0.21 0.49 0.05 0.18 -0.16

0.420 0.052 <0.0001 0.679 0.112 0.144

Other fungal

parasitism

0.55 -0.11 -0.44 -0.07 -0.31

<0.0001 0.300 <0.0001 0.527 0.004

Bacterial

parasitism

0.05 -0.73 0.14 -0.52

0.672 <0.0001 0.217 <0.0001

Total

parasitism*

0.18 0.10 0.04

0.108 0.388 0.699

Soil pH

0.10 0.67

0.360 <0.0001

Ceh

0.09

0.442

Post soil

moisture

*Total parasitism including zygomycetes, other fungi, and bacteria without Trichoderma

@Each top row within each variable represents spearman correlation coefficients and bottom row

represents the p value. Correlation is significant at p<0.05 level. Bold text represents significant

p value.

183

Figure 5-1. Pot set-up in the growth chamber for ASD study on beneficial organism.

Pot set-up with oxidation-reduction electrode and pathogen bags (A-B), pots saturated with

deionized water (or deionized water with Trichoderma spore suspension), covered with

polyethylene (C), ASD termination at 3 wks.(D), bag removal, from 5-, 10- and 15-cm depths

(E), surface-sterilization and plating of sclerotia onto plate well plate (F), location of sclerotial

bag in the pots (G), sclerotia plated on respective media (H), and observation of sclerotial

germination and parasitism by parasites.

G. Sclerotial bag location I. Sclerotial germination & parasitism

PDA+rif

Actinomycetes

Isolation Agar

Trichoderma

Selective Media

10 cm

15 cm

5 cm

H. Sclerotia plated on media

A EDCB F

184

Figure 5-2. Process of soil serial dilutions to measure actinomycetes, Bacillus and Trichoderma

populations.

Soil serial

Diluation

Actinomycetes

Bacillus

Trichoderma

185

Figure 5-3. Effect of ASD (with/without biofungicides) and biofungicides on (A) cumulative

anaerobic condition, (B) soil pH, and (C) gravimetric soil moisture content.


PLSD test. Error bars indicate standard error with four replicates.

186

de

cde

cd

cd

e

ab

b

bc

a

ab

ab

0 50 100

ASD + T. asperellum

ASD + Rootshield®

ASD + Mycostop®

ASD + Mycostop® + Rootshield®

ASD

T. asperellum

Rootshield®

Mycostop®

Mycostop®+Rootshield®

Covered Ctrl

Non-covered Ctrl

Sclerotium germination at 5 cm (%)

AA

ASD

Non ASD

Control

c

c

b

c

c

a

ab

ab

ab

ab

ab

*

0 50 100

ASD+T. asperellum

ASD+Rootshield®

ASD+Mycostop®

ASD+Mycostop®+Rootshield®

ASD

T. asperellum

Rootshield®

Mycostop®


Covered Ctrl

Non-covered Ctrl

Lab CtrlSclerotial germination at 10 cm (%)

ASD

Non ASD

Control

B

bc

d

d

d

cd

b

cd

a

b

a

b

0 50 100

ASD+T. asperellum

ASD+Rootshield®

ASD+Mycostop®


ASD

T. asperellum

Rootshield®

Mycostop®


Covered Ctrl

Non-covered Ctrl

Sclerotial germination at 15cm (%)

ASD

Non ASD

Control

C

A

Figure 5-4. Effect of ASD (with/without biofungicides) and biofungicides on percentage

sclerotial germination from sclerotia recovered from (A) 5-cm examined on PDA, (B) 10-cm

examined on AIA, and (C) 15-cm examined on TSM.



187

Figure 5-5.Effect of ASD (with/without biofungicides) and biofungicides on (A) percentage

sclerotial parasitism by Trichoderma, (B), zygomycetes, (C) other fungi, and (D) bacteria

examined on PDA. Sclerotia were recovered from a depth of 5-cm.


PLSD test. Error bars indicate standard error with four replicates. ASD=Pots amended with dry

molasses, T. asp.=T. asperellum, Rs=Rootshield®, Ms=Mycostop®, Ctrl=control

188


sclerotial parasitism by actinomycetes on AIA. Sclerotia were recovered from a depth of 10-cm.



e

e

a

c

de

cd

e

b

c

de

e

0 20 40 60 80 100

ASD+T. asperellum

ASD+Rootshield®

ASD+Mycostop®


ASD

T. asperellum

Rootshield®

Mycostop®


Covered Ctrl

Non-covered Ctrl

Actinomycetes parasitism of sclerotia (%)

189


sclerotial parasitism by (A) Trichoderma (B), zygomycetes (C) other fungi, and (D) bacteria on

AIA. Sclerotia were recovered from a depth of 10-cm.


PLSD test. Error bars indicate standard error with four replicates. ASD=Amended with dry


190

Figure 5-8. Effect of ASD (with/without biofungicides) and biofungicides on (A) percentage

sclerotial parasitism by Trichoderma (B), zygomycetes (C) other fungi and (D) bacteria

examined on TSM. Sclerotia were recovered from a depth of 15-cm.


PLSD test. Error bars indicate standard error with four replicates. ASD=Pots amended with dry


191

Figure 5-9. Effect of ASD (with/without biofungicides) and biofungicides on soil population of

actinomycete spp.



d

d

cd bc

ab ab

ab ab ab

a

abc

0

1

2

3

4

5

6

AS

D+

T.

as

pe

rell

um

AS

D+

Ro

ots

hie

ld®

AS

D+

My

co

sto

p®

AS

D+

My

co

sto

p®

+R

oo

tsh

ield

®

AS

D

T.

as

pere

llu

m

Ro

ots

hie

ld®

My

co

sto

p®

My

co

sto

p®

+R

oo

tsh

ield

®

Co

ve

red

Ctr

l

No

n-c

ov

ere

d C

trl

ASD Non ASD Control

Be

ne

fic

ial o

rga

nis

ms

po

pu

lati

on

(l

og

CF

U+

1)

g-1

of

so

il

Actinomycetes

192

Figure 5-10. Effect of ASD (with/without biofungicides) and biofungicides on soil populations

of Bacillus spp.



A

B

C

A

B C

C C C C C

0

1

2

3

4

5

6

7

AS

D+

T.

as

pe

rell

um

AS

D+

Ro

ots

hie

ld®

AS

D+

My

co

sto

p®

AS

D+

My

co

sto

p®

+R

oo

tsh

ield

®

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193

Figure 5-11. Effect of ASD (with/without biofungicides) and biofungicides on soil populations

of Trichoderma spp.



bcd a

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cd d d

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2

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194

Figure 5-12. Effect of ASD (with/without biofungicides) and biofungicides on cowpea nodules

number and mass.



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ab

bc bc bc bc

c

c c

bc

c

0

0.5

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t)

Total nodules no. Total nodules mass

195

Figure 5-13. Effect of amendment C:N ratio on percentage root colonization after ASD

treatment, 2014.


protected LSD test. ASD10=C:N ratio 10:1, ASD20=C:N ratio 20:1, ASD30=C:N ratio 30:1,


and UTC=Untreated, non-amended control.

.

A A AB

A

BC

C

C

0

10

20

30

40

50

60

70

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ASD10 ASD20 ASD30 ASD40 LCASD30 Fum UTC

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196

Figure 5-14. Effect of ASD (with/without biofungicides) and biofungicides on root biomass of

cowpea and tomato.



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tal

roo

t b

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as

s (

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Tomato

Cowpea

197

Figure 5-15. Effect of ASD (with/without biofungicides) and biofungicides on shoot biomass of

cowpea and tomato.



a

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5

10

15

20

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Tomato

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198

Figure 5-16. Phylogenetic analysis of major soil microbial fungi identified in ASD treated soil.

Sequences were aligned using ClustalW and phylogenetic tree was constructed using Mega 5.6.

The values in the tree represent bootstrap value for 100 replications.

199

Summary

The harmful effects of agro-chemicals on the environment, agro-biodiversity, and human beings

have been well established. Therefore, there is an increasing concern for the production methods

of horticultural products, which has motivated farmers to shift from current production practices

that utilize chemical soil fumigants to non-chemical practices. However, the lack of promising

non-chemical alternatives is a major problem for growers even if they intend to shift toward a

non-chemical approach. In addition, the phase out of the broad-spectrum fumigant methyl

bromide has compelled additional growers to seek alternatives and it is imperative that we

develop reliable non-chemical options that will control pests and maintain crop yields. Flooding,

solarization, steam sterilization, and bio-fumigation are options that can be adopted in regions

where water availability, high temperature, high investment, and site-specific crucifer production

are not limiting, respectively. For regions with any of these limitations, anaerobic soil

disinfestation (ASD) could be an alternative to management of pathogens, weeds, and nematodes

in high-value horticultural crops. However, before adopting ASD production systems, it is

imperative to optimize the conditions of ASD to fit in a given production system and prevailing

environmental conditions. In this study, we carried out meta-analysis of ASD to show that ASD

is effective against pathogens and weeds, and we identified the ideal C:N ratio and C rate of

ASD amendment that is effective to control two key soilborne pathogens (Fusarium oxysporum

and Sclerotium rolfsii) and nutsedge tubers for a moderate soil temperature regime.

Our meta-analysis study on the effectiveness of ASD on pest suppression and yield of

horticultural crops showed that ASD is effective in suppressing soilborne bacterial, oomycete,

and fungal pathogens under different environmental regimes, with various soil types, soil

temperatures, and different incubation periods of treatments, compare to non-amended controls.

Analysis of ASD effectiveness on pathogen, nematodes, and weeds across a range of amendment

types (agricultural by-products – cereal bran and crop residue, cruciferous, legume, grass, protein

by-product, manure, organic acid) demonstrated that pathogen suppression was significant.

However, due to variability in reported research, more studies on nematodes and weeds are

warranted to draw conclusive results on the overall effectiveness of ASD.

200

To evaluate the impact of organic amendment C:N ratio (dry molasses or wheat bran) and C rate

(dry molasses) on ASD treatment at moderate soil temperatures (15 to 25°C), we conducted

growth chamber, greenhouse, and field studies at the University of Tennessee. Results showed

high soil anaerobic activity in all amended treatments, regardless of amendment type and rate, in

both pot and field study and lowest soil pH at amendment C:N ratio 10:1 for pot studies.

Similarly, the survival and production of introduced yellow nutsedge tubers, survival of

inoculum of Fusarium oxysporum and Sclerotium rolfsii were lowered in all amended pots at 4

mg C g-1

of soil when compared to a non-amended control. The lowest populations of F.

oxysporum were generally observed at amendment C:N ratios of 20:1 and 30:1 for the dry

molasses amendment. However, amendment type and C:N ratio did not affect the mortality of

sclerotia and a carbon rate of 2 to 4 mg C g-1

at C:N 30:1 was optimum to control the pathogen.

ASD amendment application increased parasitism of sclerotia by Trichoderma, zygomycetes,

Aspergillus, Fusarium, actinomycetes, bacteria and other unidentified fungi. The occurrence of

these mycoparasites varied across studies and was primarily driven by soil type and isolation

media in the lab. ASD with dry molasses (4 mg C g-1

of soil) at C:N 30:1, did not alter nodule

formation by rhizobia on cowpea, and soil population of beneficial actinomycetes. In addition,

ASD enhanced populations of key fungal antagonists, Trichoderma, and bacteria, Bacillus and

total root colonization by arbuscular mycorrhizae. We also observed that use of RootShield®

and/or Mycostop® or T. asperellum during ASD treatment did not enhance sclerotial mortality.

Future research focusing on the application of antagonists as seed treatment or by soil drenching

after the termination of ASD is recommended.

As reported before, organic acid assay of soil solution from pot trials at various C:N ratios

showed that acetic and butyric are the primary organic acids generated during ASD treatment,

along with isobutyric, isovaleric, methyl butryric, and propionic acid. ASD, however, did not

show any significant improvement in tomato biomass in pot studies, we observed increased

pepper fruit yield in all ASD treatments compared to both fumigated and control treatments. Our

results showed that the higher C rate (4 mg C g-1

of soil) and a C:N ratio <30:1 gave the highest

yield, suggesting C:N ratio and C rates are important determinants for yield.

Along with the above-mentioned works on optimization of organic amendments for ASD to

control pathogens and weeds, as an effort to disseminate this technique, we also set up

201

demonstration trials of ASD in Nepal to control common plant pathogens in tomato production

areas. The trials were established at the Central Horticulture Center, Kirtipur, Nepal on May

2016 and 25 farmers from various districts were trained to use this technique. For the

demonstration, a locally available agricultural by-product (rice hulls; 1 kg m-2

) was used as the

carbon supplement in the soil and tarped after irrigation.

In short, we showed that ASD is effective for control of various plant pathogens and weeds

under controlled and partially controlled environment. We recommend testing this optimized

ASD technique on farmers’ fields to evaluate constraints in working production systems.

202

Vita

The author was born on January 1985 as the first daughter of Mr. Uttam Raj Shrestha and Mrs.

Sheela Shrestha in Kalimati-14, Kathmandu, Nepal. She pursued her Bachelor Degree in

Agricultural Science majoring in Horticulture from Tribhuvan University, Institute of

Agriculture and Animal Science, Rampur, Chitwan in 2007. After receiving her B.S. degree, she

started her Master of Science in Agriculture majoring in Conservation Ecology in 2009. She

worked as a Horticulture Development Officer for the Government of Nepal for three years and

then in 2013, she began her Doctor of Philosophy program in the Department of Plant Sciences

at the University of Tennessee, Knoxville, under the direction of Dr. David M. Butler. Her

dissertation research focused on optimization of organic amendments used in anaerobic soil

disinfestation to control key soilborne pathogens and weeds.

For this dissertation research, she was financially supported by USDA-NIFA, Southern Region -

Sustainable Agriculture Research and Education and Organic Crop Improvement Association

(OCIA) Research & Education.

anaerobic soil disinfestation: meta-analysis and

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